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WO2016160615A1 - Maytansinoid derivatives, conjugates thereof, and methods of use - Google Patents

Maytansinoid derivatives, conjugates thereof, and methods of use Download PDF

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Publication number
WO2016160615A1
WO2016160615A1 PCT/US2016/024343 US2016024343W WO2016160615A1 WO 2016160615 A1 WO2016160615 A1 WO 2016160615A1 US 2016024343 W US2016024343 W US 2016024343W WO 2016160615 A1 WO2016160615 A1 WO 2016160615A1
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WIPO (PCT)
Prior art keywords
compound
formula
integer
asparagine
antibody
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PCT/US2016/024343
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French (fr)
Inventor
Thomas Nittoli
Thomas Markotan
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Regeneron Pharmaceuticals Inc
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Regeneron Pharmaceuticals Inc
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Priority to CA2978340A priority Critical patent/CA2978340C/en
Priority to BR112017020149A priority patent/BR112017020149A8/en
Priority to KR1020247030839A priority patent/KR20240142591A/en
Priority to MX2017012380A priority patent/MX386328B/en
Priority to KR1020177030678A priority patent/KR102708103B1/en
Priority to NZ735426A priority patent/NZ735426B2/en
Priority to CN201680030443.2A priority patent/CN107995912A/en
Priority to EP16714199.3A priority patent/EP3273998B1/en
Priority to EA201700464A priority patent/EA034950B1/en
Priority to MA41065A priority patent/MA41065B1/en
Priority to JP2017550548A priority patent/JP6948950B2/en
Priority to SG11201707148PA priority patent/SG11201707148PA/en
Application filed by Regeneron Pharmaceuticals Inc filed Critical Regeneron Pharmaceuticals Inc
Priority to CN202210829028.0A priority patent/CN115645543A/en
Priority to AU2016243527A priority patent/AU2016243527B2/en
Publication of WO2016160615A1 publication Critical patent/WO2016160615A1/en
Priority to IL254267A priority patent/IL254267B/en
Priority to ZA2017/06040A priority patent/ZA201706040B/en
Priority to PH12017501780A priority patent/PH12017501780A1/en
Anticipated expiration legal-status Critical
Priority to CONC2017/0010890A priority patent/CO2017010890A2/en
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5365—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68033—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a maytansine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/08—Bridged systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
    • C07D498/18—Bridged systems

Definitions

  • FIELD FIELD
  • the present disclosure concerns maytansinoid derivatives, conjugates thereof, and methods of treating or preventing proliferative diseases with the same.
  • BACKGROUND Proliferative diseases, for example cancer, are characterized by the uncontrolled growth of abnormal cells. Current treatments of proliferative diseases include surgery, radiation, chemotherapy, hormone-based therapy and/or immunotherapy. A number of these treatments, particularly chemotherapy, utilize anti-proliferative drugs that limit the spread of the abnormal cells. However, these drugs are typically indiscriminate in their ability to kill cells, affecting both normal and abnormal cells.
  • ADCs Antibody drug conjugates
  • ADCs are compounds composed of an antibody that is linked, via a chemical linker, to a cytotoxic agent. Such compounds leverage the antibody’s binding specificity for its target to deliver a cytotoxic agent to an abnormal cell.
  • A is arylene or heteroarylene
  • L is a linker
  • BA is a binding agent
  • k is an integer from 1 to 30. Also provided herein are stereoisomers of compounds of Formula (I).
  • A is arylene or heteroarylene.
  • PT1 or a salt thereof, wherein A is arylene or heteroarylene and L is a linker. Also provided herein are stereoisomers of compounds of Formula PT1.
  • kits for treating proliferative diseases comprising administering the compounds described herein.
  • methods of treating proliferative diseases comprising administering the conjugates described herein.
  • methods of preparing compounds of Formula (I) comprising reacting a deglycosylated antibody or aglycosylated antibody with a compound of Formula (PT1) in the presence of transglutaminase.
  • PT1 a compound of Formula (PT1) in the presence of transglutaminase.
  • FIG 2 depicts the plot of % Cell Viability vs. Log 10 [M] of certain compounds tested in EXAMPLE 41.
  • FIG 3 depicts the plot of % Cell Viability vs. Log 10 [M] of certain compounds tested in EXAMPLE 41.
  • FIG.4 depicts the plot of % Cell Viability vs. Log 10 [M] of certain compounds tested in EXAMPLE 41.
  • FIG.5 depicts the plot of % Cell Viability vs. Log 10 [M] of certain compounds tested in EXAMPLE 41.
  • FIG.6 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(4-amino-2-fluoro)benzamido-Cit-Val-Cap-Mal.
  • FIG.7 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(4-amino-2-trifluoromethyl)benzamido-Cit-Val-Cap-Mal.
  • FIG.8 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(4-amino-2-methoxy)benzamido-Cit-Val-Cap-Mal.
  • FIG.9 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-4-aminobenzamide.
  • FIG.10 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(2-fluoro-4-amino)benzamide
  • FIG.11 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(2-trifluoromethyl-4-amino)benzamide.
  • FIG.12 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(2-methoxy-4-amino)benzamide.
  • FIG.13 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(3-trifluoromethyl-4-amino)benzamide.
  • FIG.14 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(2-chloro-4-amino-5-fluoro)benzamide.
  • FIG.15 depicts a general synthetic sequence for preparing compounds of Formula (II) wherein substituent R is defined herein and below.
  • FIG.16 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(2,5-difluoro-4-amino)benzamide.
  • FIG.17 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(3-fluoro-4-amino)benzamide alanine-(3-chloro-4-amino)benzamide.
  • FIG.19 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(5-amino-8-carboxyquinoline)carboxamide.
  • FIG.20 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(3-bromo-4-amino)benzamide.
  • FIG.21 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(3-methoxy-4-amino)benzamide.
  • FIG.22 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(2-methyl-4-amino)benzamide.
  • FIG.23 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(3-methyl-4-amino)benzamide.
  • FIG.24 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(8-amino-5-carboxyquinoline)carboxamide.
  • FIG.25 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(3-methoxy-4-amino)benzamido-Cit-Val-Cap-Mal.
  • FIG.26 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(2-fluoro-4-amino)benzamido-Cit-Val-Cap-6-amine.
  • FIG.27 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(2-methoxy-5-amino)benzamide.
  • FIG.28 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(3-amino-4-methoxy)benzamide.
  • FIG.29 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(3-amino-5-fluoro)benzamide.
  • FIG.30 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(2-fluoro-5-amino)benzamide.
  • FIG.31 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(3-amino)benzamide.
  • FIG.32 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(3-amino-4-fluoro)benzamide.
  • FIG.33 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-4-aminobenzamide-adipic-NHS.
  • FIG.34 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-4-aminobenzamide-Cap-Mal. alanine-N-(3-methylsulfonyl-4-amino)benzamide.
  • FIG.36 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(3-hydroxy-4-amino)benzamide.
  • FIG.37 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(2-amino)benzamide.
  • FIG.38 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(4-methoxy-2-amino)benzamide.
  • FIG.39 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(3-morpholino-4-amino)benzamide.
  • FIG.40 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(3-acetamido-4-amino)benzamide.
  • FIG.41 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-4-aminobenzamide-Cit-Val-cap-diBromomethylacryl.
  • FIG.42 depicts the deconvoluted mass spectroscopy (MS) spectrum of the antibody drug conjugate, PRLR-Q-63 conjugate from EXAMPLE 43.
  • FIG.43 depicts the deconvoluted MS spectrum of the Isotype Control-Q-63 conjugate from EXAMPLE 43.
  • FIG.44 depicts the plot of % Cell Viability vs. Log 10 [M] of certain compounds tested in EXAMPLE 45.
  • FIG.45 depicts the plot of % Cell Viability vs. Log 10 [M] of certain compounds tested in EXAMPLE 45.
  • FIG.46 depicts the plot of % Cell Viability vs. Log10 [M] of certain compounds tested in EXAMPLE 45.
  • FIG.47 depicts the plot of % Cell Viability vs. Log10 [M] of certain compounds tested in EXAMPLE 45.
  • alkyl refers to a monovalent and saturated hydrocarbon radical moiety. Alkyl is optionally substituted and can be linear, branched, or cyclic, i.e., cycloalkyl.
  • Alkyl includes, but is not limited to, those having 1-20 carbon atoms, i.e., C 1-20 alkyl; 1-12 carbon atoms, i.e., C 1-12 alkyl; 1-8 carbon atoms, i.e., C 1-8 alkyl; 1-6 carbon atoms, are not limited to methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, i-butyl, a pentyl moiety, a hexyl moiety, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
  • haloalkyl refers to alkyl, as defined above, wherein the alkyl includes at least one substituent selected from a halogen, e.g., F, Cl, Br, or I.
  • alkenyl refers to a monovalent hydrocarbon radical moiety containing at least two carbon atoms and one or more non-aromatic carbon-carbon double bonds. Alkenyl is optionally substituted and can be linear, branched, or cyclic.
  • Alkenyl includes, but is not limited to, those having 2-20 carbon atoms, i.e., C 2-20 alkenyl; 2-12 carbon atoms, i.e., C 2-12 alkenyl; 2-8 carbon atoms, i.e., C 2-8 alkenyl; 2-6 carbon atoms, i.e., C 2-6 alkenyl; and 2-4 carbon atoms, i.e., C 2-4 alkenyl.
  • alkenyl moieties include, but are not limited to vinyl, propenyl, butenyl, and cyclohexenyl.
  • alkynyl refers to a monovalent hydrocarbon radical moiety containing at least two carbon atoms and one or more carbon-carbon triple bonds. Alkynyl is optionally substituted and can be linear, branched, or cyclic.
  • Alkynyl includes, but is not limited to, those having 2-20 carbon atoms, i.e., C 2-20 alkynyl; 2-12 carbon atoms, i.e., C 2-12 alkynyl; 2-8 carbon atoms, i.e., C 2-8 alkynyl; 2-6 carbon atoms, i.e., C 2-6 alkynyl; and 2-4 carbon atoms, i.e., C 2-4 alkynyl.
  • alkynyl moieties include, but are not limited to ethynyl, propynyl, and butynyl.
  • alkoxy refers to a monovalent and saturated hydrocarbon radical moiety wherein the hydrocarbon includes a single bond to an oxygen atom and wherein the radical is localized on the oxygen atom ⁇ e.g. ⁇ CH 3 CH 2 -O ⁇ for ethoxy.
  • Alkoxy substituents bond to the compound which they substitute through this oxygen atom of the alkoxy substituent.
  • Alkoxy is optionally substituted and can be linear, branched, or cyclic, i.e., cycloalkoxy.
  • Alkoxy includes, but is not limited to, those having 1-20 carbon atoms, i.e., C 1-20 alkoxy; 1-12 carbon atoms, i.e., C 1-12 alkoxy; 1-8 carbon atoms, i.e., C 1-8 alkoxy; 1-6 carbon atoms, i.e., C 1-6 alkoxy; and 1-3 carbon atoms, i.e., C 1-3 alkoxy.
  • alkoxy moieties include, but are not limited to methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s- butoxy, t-butoxy, i-butoxy, a pentoxy moiety, a hexoxy moiety, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy.
  • haloalkoxy refers to alkoxy, as defined above, wherein the alkoxy includes at least one substituent selected from a halogen, e.g., F, Cl, Br, or I.
  • aryl refers to a monovalent moiety that is a radical of an aromatic compound wherein the ring atoms are carbon atoms.
  • Aryl is optionally substituted include, but are not limited to those having 6 to 20 ring carbon atoms, i.e., C 6-20 aryl; 6 to 15 ring carbon atoms, i.e., C 6-15 aryl, and 6 to 10 ring carbon atoms, i.e., C 6-10 aryl.
  • Examples of aryl moieties include, but are limited to phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, and pyrenyl.
  • arylene refers to a divalent moiety of an aromatic compound wherein the ring atoms are only carbon atoms.
  • Arylene is optionally substituted and can be monocyclic or polycyclic, e.g., bicyclic or tricyclic.
  • aryl moieties include, but are not limited to those having 6 to 20 ring carbon atoms, i.e., C 6-20 arylene; 6 to 15 ring carbon atoms, i.e., C 6-15 arylene, and 6 to 10 ring carbon atoms, i.e., C 6-10 arylene.
  • alkaryl refers to an aryl that is substituted with at least one alkyl. Alkaryl is optionally substituted.
  • heteroalkyl refers to an alkyl in which one or more carbon atoms are replaced by heteroatoms.
  • heteroalkenyl refers to an alkenyl in which one or more carbon atoms are replaced by heteroatoms.
  • heteroalkynyl refers to an alkenyl in which one or more carbon atoms are replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen, oxygen, and sulfur atoms. Heteroalkyl is optionally substituted. Examples of heteroalkyl moieties include, but are not limited to, aminoalkyl, sulfonylalkyl, sulfinylalkyl. Examples of heteroalkyl moieties also include, but are not limited to, methylamino, methylsulfonyl, and methylsulfinyl.
  • heteroaryl refers to a monovalent moiety that is a radical of an aromatic compound wherein the ring atoms contain carbon atoms and at least one oxygen, sulfur, nitrogen, or phosphorus atom.
  • heteroaryl moieties include, but are not limited to those having 5 to 20 ring atoms; 5 to 15 ring atoms; and 5 to 10 ring atoms.
  • Heteroaryl is optionally substituted.
  • heteroarylene refers to an arylene in which one or more ring atoms of the aromatic ring are replaced with an oxygen, sulfur, nitrogen, or phosphorus atom. Heteroarylene is optionally substituted.
  • heterocycloalkyl refers to a cycloalkyl in which one or more carbon atoms are replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen, oxygen, and sulfur atoms. Heterocycloalkyl is optionally substituted. Examples of heterocycloalkyl moieties include, but are not limited to, morpholinyl, piperidinyl, dithiolanyl, oxanyl, or thianyl.
  • optionally substituted when used to describe a radical moiety, e.g., optionally substituted alkyl, means that such moiety is optionally bonded to one or more substituents.
  • substituents include, but are not limited to halo, cyano, nitro, haloalkyl, azido, epoxy, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, , , , , , , , or , wherein R A , R B , and R C are, independently at each occurrence, a hydrogen atom, alkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl, heteroalkyl, heteroaryl, or heterocycloalkyl, or R A and R B , together with the atoms to which they are bonded, form a saturated or unsaturated carbocyclic ring, wherein the ring is optionally substituted
  • R A is methylamino.
  • R A is methylamino.
  • the substituents on the optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted saturated or unsaturated carbocyclic ring, if they are substituted, are not substituted with substituents which are further optionally substituted with additional substituents.
  • the substituent bonded to the group is unsubstituted unless otherwise specified.
  • binding agent refers to any molecule capable of binding with specificity to a given binding partner.
  • linker refers to a divalent moiety that covalently links the binding agent to the maytansinoid derivatives described herein.
  • amide synthesis conditions refers to reaction conditions suitable to effect the formation of an amide, e.g., by the reaction of a carboxylic acid, activated carboxylic acid, or acyl halide with an amine.
  • amide synthesis conditions refers to reaction conditions suitable to effect the formation of an amide bond first converted to an activated carboxylic acid before the activated carboxylic acid reacts with an amine to form an amide.
  • Suitable conditions to effect the formation of an amide include, but are not limited to, those utilizing reagents to effect the reaction between a carboxylic acid an amine, including, but not limited to, dicyclohexylcarbodiimide (DCC),
  • DCC dicyclohexylcarbodiimide
  • HATU hexafluorophosphate
  • EEDQ 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline
  • EDC 1- Ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • CIP 2-Chloro-1,3- dimethylimidazolidinium hexafluorophosphate
  • CDMT 2-chloro-4,6-dimethoxy-1,3,5-triazine
  • CDI carbonyldiimidazole
  • a carboxylic acid is first converted to an activated carboxylic ester before reacting with an amine to form an amide bond.
  • the carboxylic acid is reacted with a reagent.
  • the reagent activates the carboxylic acid by deprotonating the carboxylic acid and then forming a product complex with the deprotonated carboxylic acid as a result of nucleophilic attack by the deprotonated carboxylic acid onto the protonated reagent.
  • this activated ester is more susceptible subsequently to nucleophilic attack by an amine than the carboxylic acid is before it is converted. This results in amide bond formation.
  • the carboxylic acid is described as activated.
  • Exemplary reagents include DCC and DIC.
  • terapéuticaally effective amount refers to an amount (of a compound) that is sufficient to provide a therapeutic benefit to a patient in the treatment or management of a disease or disorder, or to delay or minimize one or more symptoms associated with the disease or disorder.
  • heterocycloalkyl through a bond between ring atoms are meant to indicate, unless specified otherwise, that the cyclic group may be substituted with that substituent at any ring position in the cyclic group or on any ring in the fused ring group, according to techniques set forth herein or which are known in the field to which the instant disclosure pertains. For example,
  • the group, in which the positions of substituents other than R 1 which are bonded to the cyclic group through a bond between ring atoms are described generically, includes the following groups in which the positions of these substituents other than R 1 are described specifically:
  • the substituent R 1 may be bonded to any ring position in the cyclic group or on any ring in the fused ring group which is not occupied by one of these substituents other than R 1 .
  • the following non-limiting representative illustrations indicate that the cyclic group can be substituted with the indicated substituent at any ring position or ,
  • A is arylene or heteroarylene
  • L is a linker
  • BA is a binding agent
  • A is arylene. In some embodiments, A is
  • the arylene or heteroarylene is substituted with one or more electron withdrawing groups and/or one or more electron donating groups.
  • A is a divalent radical of benzene, of pyridine, of naphthalene, or of quinolone, which are optionally substituted. substituted with a member selected from the group consisting of amino, amido, alkyl, halo, haloalkyl, alkoxy, and haloalkoxy.
  • A is:
  • R 1 independently at each occurrence, is alkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl, halo, heteroaryl, heterocycloalkyl, hydroxyl, cyano, nitro, , , , or azido,
  • R A is alkyl or heteroalkyl
  • n is an integer from 0 to 4.
  • n is an integer from 0 to 3;
  • p is an integer from 0 to 6;
  • q is an integer from 0 to 5.
  • A is:
  • R 1 is, independently at each occurrence, halo, haloalkyl, haloalkoxy, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, aryl, alkaryl, aralkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitr , or azido,
  • R A is alkyl o
  • n is an integer from 0 to 4.
  • n is an integer from 0 to 3;
  • p is an integer from 0 to 6;
  • q is an integer from 0 to 5.
  • A is: wherein:
  • R 1 is, independently at each occurrence, halo, haloalkyl, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitro , or azido,
  • n is an integer from 0 to 4.
  • n is an integer from 0 to 3;
  • p is an integer from 0 to 6;
  • q is an integer from 0 to 5.
  • R 1 is C 1-6 alkyl or C 1-6 alkoxy. In some of these embodiments, R 1 is methyl, ethyl, methoxy, or ethoxy. In some of these embodiments, R 1 is methoxy.
  • R 1 is, independently, alkyl or halo. In some embodiments, R 1 is, independently, C 1-6 alkyl, C 1-6 haloalkyl, or halo. In some embodiments, R 1 is, independently, halo. In some embodiments, R 1 is, independently, fluoro, chloro, bromo, iodo, or trifluoromethyl. In some embodiments, n, m, p, or q is 0, 1 or 2. In some embodiments, n, m, p, or q is 0 or 1. In some embodiments, n, m, p, or q is 0.
  • R 1 is . In some embodiments, R 1 is wherein R A is methyl. In some embodiments, R 1 is hydroxyl. In some embodiments, R 1 is N- methylformamide. In some embodiments, R 1 is morpholinyl.
  • R 1 is, independently, alkyl, alkoxy, or halo. In some embodiments, R 1 is, independently, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 haloalkoxy, or halo. In some embodiments, R 1 is, independently, halo. In some embodiments, R 1 is, independently, fluoro, chloro, bromo, iodo, or trifluoromethyl. In some embodiments, n, m, p, or q is 0, 1 or 2. In some embodiments, n, m, p, or q is 0 or 1. In some embodiments, n, m, p, or q is 0.
  • A is: .
  • A A .
  • n 0, 1 or 2.
  • A is:
  • n 0, 1 or 2.
  • A is:
  • n is 0 or 1; and R 1 is alkoxy, halo, or haloalkyl.
  • A is:
  • n is 0 or 1; and R 1 is alkoxy, ha alkyl. In some examples, R 1 is alkoxy.
  • A is: ,
  • n is 0 or 1; and R 1 is C 1-6 alk 1-6 haloalkyl.
  • a s [0096] In some embodiments, A s:
  • n is 0 or 1; and R 1 is C 1-6 alkox C 1-6 haloalkyl. In some examples, R 1 is C 1-6 alkoxy.
  • A is:
  • b is an integer from 2 to 8 and is a
  • A is:
  • A is:
  • n 0, 1, 2, 3, or 4.
  • A is:
  • n 0, 1, 2, 3, or 4.
  • R 1 is C
  • n 0, 1 or 2.
  • A is:
  • R 1 is C 1-6 alkyl, halo, or C 1-6 haloalkyl
  • n 0, 1 or 2.
  • A is:
  • R 1 is C 1-6 alkyl, C 1-6 l, or C 1-6 haloalkoxy
  • n 0, 1, 2, 3, or 4.
  • A is: ,
  • R 1 is C 1-6 alkyl, C 1-6 alkoxy, halo, C 1-6 haloalkyl, or C 1-6 haloalkoxy; and n is 0, 1, 2, 3, or 4.
  • A is:
  • R 1 is C 1-6 alkyl, C 1-6 alkoxy, aloalkyl. In certain of these embodiments, R 1 is methoxy or methyl. In some specific embodiments, R 1 is methoxy.
  • A is: .
  • A is:
  • R 1 is halo or trifluoromethyl
  • n 0, 1 or 2.
  • A is: .
  • X is a hydrogen atom, halo, or trifluoromethyl.
  • A is: wherein:
  • X is a hydrogen atom, halo, or trifluoromethyl; is the bond to the nitrogen atom; and is the bond to the carbonyl.
  • A is:
  • R 1 is, independently at each occurrence, a hydrogen atom, alkyl, alkoxy, aryl, heteroalkyl, halo, haloalkyl, haloalkoxy or hydroxyl; is the bond to the nitrogen atom; and is the bond to the carbonyl.
  • R 1 is 1-methylethyl- iol, phenyl, 2-fluorophenyl, pyridinyl, 4-pyridinyl, pyrrolidinyl, or 1- pyrrolidinyl.
  • R 1 is trifluoromethyl.
  • R 1 is methoxy.
  • R 1 is fluoro.
  • R 1 is hydrogen.
  • A is:
  • R 1 is, independently at each occurrence, a hydrogen atom, alkyl, alkoxy, aryl, heteroalkyl, halo, haloalkyl, haloalkoxy, or hydroxyl; is the bond to the nitrogen atom; and is the bond to the carbonyl.
  • R 1 is 1-methylethyl- thiol, phen yl, luorophenyl, pyridinyl, 4-pyridinyl, pyrrolidinyl, or 1-pyrrolidinyl.
  • R 1 is trifluoromethyl.
  • R 1 is methoxy.
  • R 1 is fluoro.
  • R 1 is hydrogen. methylformamide.
  • R 1 is hydroxyl.
  • R 1 is morpholinyl.
  • A is:
  • R 1 is, independently at each occurrence, a hydrogen atom, alkyl, alkoxy, aryl, heteroalkyl, halo, haloalkyl, or haloalkoxy; is the bond to the nitrogen atom; and is the bond to the carbonyl.
  • R 1 is alkyl or alkoxy.
  • R 1 is propylamino, difluoro-methoxy, phenyl, 2-fluorophenyl.
  • R 1 is trifluoromethyl. In some embodiments, R 1 is methoxy. In some embodiments, R 1 is fluoro. In some embodiments, R 1 is hydrogen.
  • A is:
  • A is:
  • A is:
  • A is:
  • A is:
  • X is F, Cl, Br, CN, methoxy, dimethylamino or cyclopropyl; is the bond to the nitrogen atom; and is the bond to the carbonyl.
  • A is:
  • X is F, Cl, Br, CN, methoxy, dimethylamino, 1-methyl-ethyl-thio or cyclopropyl; is the bond to the nitrogen atom; and is the bond to the carbonyl.
  • A is: ,
  • each R 1 is independently, at e ce, a hydrogen atom, alkyl, alkoxy, halo, haloalkyl, or haloalkoxy; is the bond to the nitrogen atom; and is the bond to the carbonyl.
  • R 1 is hydrogen, fluoro, trifl omethyl, or methoxy.
  • R 1 is fluoro, chloro, bromo, or iodo.
  • A is:
  • A is:
  • each R 1 is independently, at e e, a hydrogen atom, alkyl, alkoxy, halo, haloalkyl, or haloalkoxy, is the bond to the nitrogen atom; and is the bond to the carbonyl.
  • A is:
  • A is:
  • A is:
  • a d is the bond to the carbonyl.
  • A is:
  • A is:
  • A is: ; is the bond to the and is the bond to the carbonyl.
  • A is:
  • A is:
  • A is:
  • A is the bond to the carbonyl.
  • A is the bond to the nitrogen atom; and is the bond to the carbonyl.
  • A is the bond to the nitrogen atom; and is the bond to the carbonyl.
  • A is the bond to the nitrogen atom; and is the bond to the carbonyl.
  • A is:
  • X s,nepenenty at eac occurrence, a yrogen atom, a y, a oxy, halo, haloalkoxy, haloalkyl, heteroalkyl.
  • X is fluoro, chloro, bromo, iodo, dimethylamino, methylamino, methoxy, ethoxy, or trifluoromethyl.
  • A is: 1 1 , X
  • X is, independently at each occurrence, a hydrogen atom, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, heteroalkyl.
  • X is fluoro, chloro, bromo, iodo, dimethylamino, methylamino, methoxy, ethoxy, trifluoromethyl or methoxy;
  • linker portion of the conjugates described herein is a divalent moiety that covalently links the binding agent to the maytansinoid derivatives described herein. Suitable linkers include those that release the maytansinoid portion in the presence of an enzyme or at a particular pH range or value.
  • the linker comprises an enzyme-cleavable moiety.
  • Illustrative enzyme-cleavable moieties include, but are not limited to, peptide bonds, ester linkages, hydrazones, and disulfide linkages.
  • the linker comprises a cathepsin-cleavable linker.
  • the linker comprises a non-cleavable moiety.
  • the non-cleavable linker i or a residue thereof In some embodiments, the non-cleavab or a residue thereof.
  • Suitable linkers also include, but are not limited to, those that are chemically bonded to two cysteine residues of a single binding agent, e.g., antibody. Such linkers can serve to mimic the antibody’s disulfide bonds that are disrupted as a result of the conjugation process.
  • the linker comprises one or more amino acids. Suitable amino acids include natural, non-natural, standard, non-standard, proteinogenic, non- proteinogenic, and L-, or D- ⁇ ⁇ amino acids.
  • the linker comprises alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, or derivative thereof.
  • the linker comprises valine and citrulline.
  • the linker is:
  • SP is a spacer
  • a 1 is an amino acid
  • AA 2 is an amino acid.
  • the spacer is a divalent moiety that connects the AA 1 -AA 2 moiety to the binding agent (BA).
  • Suitable spacers include, but are not limited to, those comprising alkylene or polyethylene glycol.
  • the ends of the spacers, i.e., the portion of the spacer directly bonded to the binding agent or AA 1 can be moieties derived from reactive moieties that are used for purposes of coupling the naked antibody or AA 1 to the spacer during the chemical synthesis of the conjugate.
  • suitable spacers include, but are not limited to, a primary amine-terminated alkylene or a primary amine-terminated polyethylene glycol.
  • the primary amine-terminating end of the spacer can be directly bonded to a deglycosylated antibody or aglycosylated antibody in the presence of transglutaminase.
  • the spacer comprises a C 5-7 alkylene. In some embodiments, the spacer is:
  • b is an integer from 2 to 8.
  • the spacer comprises a primary amine-terminated alkylene. In some embodiments, the spacer comprises a NH 2 -C 5-7 alkylene. In some embodiments, the spacer is:
  • the spacer is:
  • the spacer is:
  • R N is a hydrogen atom or alkyl
  • R M is alkyl; the two bonds represented by are bonds to cysteines of a binding agent;
  • b is an integer from 2 to 8.
  • the spacer is:
  • the spacer is:
  • g is 2-8. In some
  • g is 2, 4, 6, or 8.
  • the spacer i wherein n is an integer from 4 to 10. In some embodiments, n is 4, 5, 6, [0165] In some embodim , wherein n is an integer from 4 to 10. In some embodime
  • the spacer In some embodiments, the spacer .
  • the spacer is
  • X is N or O; R N and R M are each, independently, hydrogen or alkyl; and b is an integer from 1 to 8.
  • AA 1 -AA 2 is: valine-citrulline, citrulline-valine, lysine- phenylalanine, phenylalanine-lysine, valine-asparagine, asparagine-valine, threonine- asparagine, asparagine-threonine, serine-asparagine, asparagine-serine, phenylalanine- asparagine, asparagine-phenylalanine, leucine-asparagine, asparagine-leucine, isoleucine- asparagine, asparagine-isoleucine, glycine-asparagine, asparagine-glycine, glutamic acid- asparagine, asparagine-glutamic acid, citrulline-asparagine, asparagine-citrulline, alanine- asparagine, or asparagine-alanine.
  • AA 1 -AA 2 is: valine-citrulline or citrulline-valine. In some embodiments, AA 1 -AA 2 is: valine-citrulline.
  • the linker is:
  • SP is a spacer
  • amino acid side chains include, but are not limited to, the ⁇ -carbon substituent of alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, and citrulline.
  • the linker is:
  • the linker is:
  • BA is an antibody and the linker is:
  • the two bonds represented b are bonds to cysteines of the antibody; and b is an integer from 2 to 8.
  • BA i an antibody and the linker is:
  • the two bonds represented by are bonds to cysteines of the antibody; and b is an integer from 2 to 8
  • BA is an antibody and the linker is:
  • R N is a hydrogen atom or alkyl
  • R M is alkyl; b is an integer from 2 to 8.
  • the li nker is:
  • the linker is:
  • g is an integer from 2 to 20. In some embodiments, g is 2 to 8. In some
  • g is 2, 4, 6, or 8.
  • the linker is:
  • the linker is:
  • Suitable binding agents include, but are not limited to, antibodies,
  • lymphokines hormones, growth factors, viral receptors, interleukins, or any other cell binding or peptide binding molecules or substances.
  • the binding agent is an antibody. In some embodiments, the binding agent is an antibody.
  • the antibody is a monoclonal antibody, polyclonal antibody, antibody fragment (Fab, Fab’, and F(ab)2, minibody, diabody, tribody, and the like), or bispecific antibody.
  • Antibodies herein can be humanized using methods described in US Patent No.6,596,541 and US Publication No.2012/0096572, each incorporated by reference in their entirety.
  • the binding agent binds to an antigen binding partner that is a polypeptide and may be a transmembrane molecule (e.g., receptor) or a growth factor that might be glycosylated or phosphorylated.
  • an antigen binding partner that is a polypeptide and may be a transmembrane molecule (e.g., receptor) or a growth factor that might be glycosylated or phosphorylated.
  • antigens include, but are not limited to, molecules such as renin; a growth hormone, including human growth hormone and stimulating hormone; lipoproteins; alpha1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor vmc, factor IX, tissue factor (TF), and von Willebrands factor; anti- clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta;
  • enkephalinase RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MlP-I-alpha); a serum albumin, such as human serum albumin; Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;
  • mouse gonadotropin-associated peptide such as prorelaxin; mouse gonadotropin-associated peptide; a microbial protein, such as
  • a cytotoxic T-lymphocyte associated antigen such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; protein A or D; rheumatoid factors; a neurotrophic factor such as bone- derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4, NT-5, or NT- 6), or a nerve growth factor such as NGF- ⁇ ; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; fibroblast growth factor receptor 2 (FGFR2), epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF- ⁇ 1, TGF- ⁇ 2, TGF- ⁇ 3, TGF- ⁇ 4, or TGF- ⁇ 5; insulin-like growth factor-l and -I
  • CTL cytotoxic T-lymphocyte associated antigen
  • Exemplary antigens also include, but are not limited to, BCMA, SLAMF7, B7H4, GPNMB, UPK3A, and LGR5.
  • the antigens include prolactin receptor (PRLR) or prostate-specific membrane antigen (PSMA).
  • PRLR prolactin receptor
  • PSMA prostate-specific membrane antigen
  • Binding agents also include, but are not limited to, ankyrin repeat proteins, interferons, lymphokines such as IL-2 or IL-3, hormones like insulin and glucocorticoids, growth factors such as EGF, transferrin and fibronectin type III.
  • the binding agents interact with or bind to tumor antigens, including antigens specific for a type of tumor or antigens that are shared, overexpressed or modified on a particular type of tumor.
  • tumor antigens include, but are not limited to: alpha-actinin-4 with lung cancer, ARTC1 with melanoma, BCR-ABL fusion protein with chronic myeloid leukemia, B-RAF, CLPP or Cdc27 with melanoma, CASP-8 with squamous cell carcinoma, and hsp70-2 with renal cell carcinoma as well as the following shared tumor-specific antigens, for example: BAGE-1, GAGE, GnTV, KK-LC-1, MAGE-A2, NA88-A, TRP2-INT2.
  • the binding agent is an antibody. In some embodiments, the binding agent is an antibody.
  • the binding agent is a monoclonal antibody. In some embodiments, the binding agent is a polyclonal antibody. In some embodiments, the antibody is an anti-PSMA, anti-MUC16, or anti-EGFRvIII, or anti-STEAP-2 antibody. binding molecule, through an attachment at a particular amino acid within the antibody or antigen-binding molecule. Exemplary amino acid attachments that can be used in the context of this aspect of the disclosure include, e.g., lysine (see, e.g., US 5,208,020; US
  • Linkers can be conjugated via glutamine via transglutaminase-based chemo-enzymatic conjugation (see, e.g., Dennler et al., Bioconjugate Chem.2014, 25, 569-578).
  • Linkers can also be conjugated to an antigen-binding protein via attachment to carbohydrates (see, e.g., US 2008/0305497, WO 2014/065661, and Ryan et al., Food & Agriculture Immunol., 2001, 13:127-130) and disulfide linkers (see, e.g., WO 2013/085925, WO 2010/010324, WO 2011/018611, WO 2014/197854, and Shaunak et al., Nat. Chem. Biol., 2006, 2:312-313).
  • the binding agent is an antibody, and the antibody is bonded to the linker through a lysine residue. In some embodiments, the antibody is bonded to the linker through a cysteine residue.
  • A is:
  • R 1 independently at each occurrence, is alkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl, halo, heteroaryl, heterocycloalkyl, hydroxyl, cyano, nitro, , , , or azido,
  • n is an integer from 0 to 4.
  • n is an integer from 0 to 3;
  • p is an integer from 0 to 6;
  • q is an integer from 0 to 5;
  • SP is a spacer
  • AA 1 is an amino acid
  • AA 2 is an amino acid.
  • A is:
  • R 1 independently at each occurrence, is C 1-6 alkyl, C 1-6 haloalkyl, or halo; n is an integer from 0 to 4;
  • n is an integer from 0 to 3;
  • p is an integer from 0 to 6;
  • q is an integer from 0 to 5;
  • SP is a spacer; is one or more bonds to the binding agent
  • a 1 is an amino acid
  • AA 2 is an amino acid. , wherein:
  • R 1 independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, haloalkoxy, heteroaryl, heteroalkyl, heterocycloalkyl, hydroxyl, cyano, nitro id
  • R A is alkyl or heteroalky
  • n is an integer from 0 to 4.
  • n is an integer from 0 to 3;
  • p is an integer from 0 to 6;
  • q is an integer from 0 to 5;
  • SP is a spacer
  • a 1 is an amino acid
  • AA 2 is an amino acid
  • A is:
  • R 1 independently at each occurrence, is C 1-6 alkyl, C 1-6 haloalkyl, or halo;
  • n is an integer from 0 to 4.
  • n is an integer from 0 to 3;
  • p is an integer from 0 to 6;
  • q is an integer from 0 to 5;
  • AA 1 is an amino acid
  • AA 2 is an amino acid.
  • A is:
  • R 1 independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, hydroxyl, cyano, nitro, , , , or azido,
  • n is an intege r from 0 to 4.
  • n is an integer from 0 to 3;
  • p is an integer from 0 to 6;
  • q is an integer from 0 to 5;
  • AA 1 is an amino acid
  • AA 2 is an amino acid.
  • A is:
  • R 1 independently at each occurrence, is C 1-6 alkyl, C 1-6 haloalkyl, or halo; n is an integer from 0 to 4;
  • n is an integer from 0 to 3;
  • p is an integer from 0 to 6;
  • SP is a spacer; is one or more bonds to the binding agent
  • R 1 independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, hydroxyl, cyan , or azido, m is an nteger rom 0 to 3;
  • p is an integer from 0 to 6;
  • SP is a spacer
  • R 1 independently at each occurrence, is C 1-6 alkyl, C 1-6 haloalkyl, or halo;
  • q is an integer from 0 to 5;
  • R 1 independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitro, , , , or azido,
  • n is an integer from 0 to 4.
  • n is an integer from 0 to 3;
  • p is an integer from 0 to 6;
  • q is an integer from 0 to 5;
  • R 1 independently at each occurrence, is C 1-6 alkyl, C 1-6 haloalkyl, or halo; n is an integer from 0 to 4;
  • n is an integer from 0 to 3;
  • p is an integer from 0 to 6;
  • q is an integer from 0 to 5;
  • R 1 independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, hydroxyl cyano nitro or azido
  • n is an integer from 0 to 4.
  • n is an integer from 0 to 3;
  • p is an integer from 0 to 6;
  • q is an integer from 0 to 5;
  • A is: , wherein:
  • R 1 independently at each occurrence, is C 1-6 alkyl, C 1-6 haloalkyl, or halo; and n, m, p, and q are 0, 1, or 2; and L is
  • A is:
  • R 1 independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitro or azido
  • A is:
  • R 1 is, independently at each occurrence, is C 1-6 haloalkyl, or halo; and n is 0, 1, or 2;
  • aralkyl halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, hydroxyl cyano nitro or azido
  • R AA2 is an amino acid side chain.
  • A is:
  • R 1 is, independently at each occurrence, is halo; and n is 0, 1, or 2;
  • R 1 independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, hydroxyl, cyano, nitr , or azido,
  • n is an integer from 0 to 4.
  • b is an integer from 2 to 8.
  • A is:
  • R 1 independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, hyd l it id
  • R 1 is, independently at each occurrence, a hydrogen atom, alkyl, alkoxy, aryl, heteroalkyl, halo, haloalkoxy, haloalkyl, or haloalkoxy; is the bond to the nitrogen atom; and is the bond to the carbonyl.
  • R 1 is 1-methylethyl- luorophenyl, pyridinyl, 4-pyridinyl, pyrrolidinyl, or 1-pyrrolidinyl.
  • R 1 is trifluoromethyl. In some embodiments, R 1 is methoxy. In some embodiments, R 1 is fluoro. In some embodiments, R 1 is hydrogen.
  • A is:
  • R 1 is, independently at each occurrence, a hydrogen atom, alkyl, alkoxy, aryl, heteroalkyl, halo, haloalkyl, haloalkoxy; is the bond to the nitrogen atom; and is the bond to the carbonyl.
  • R 1 is 1-methylethyl- luorophenyl, pyridinyl, 4-pyridinyl, pyrrolidinyl, or 1-pyrrolidinyl.
  • R 1 is trifluoromethyl.
  • R 1 is methoxy.
  • R 1 is fluoro.
  • R 1 is hydrogen.
  • A is: ;
  • BA is an antibody
  • A is:
  • R 1 is, independently at each occurrence, is halo; and n is 0, 1, or 2;
  • A is:
  • R 1 independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitr , or azido; and
  • SP is a spacer; is the one or more bonds to the binding agent
  • R AA1 is an amino acid side chain
  • R AA2 is an amino acid side chain.
  • A is:
  • R 1 independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitro, , , , or azido;
  • SP is a spacer
  • AA1 is an amino acid side chain
  • R AA2 is an amino acid side chain.
  • A is:
  • R 1 independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitro , or azido;
  • b is an integer from 2 to 8.
  • A is:
  • aralkyl halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitro or azido;
  • R 1 independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitro, , , , or azido;
  • L 1 is a linker which binds to the BA through a lysine residue.
  • the subscript, k represents the number of linkers, L 1 , bonded to the BA through lysine residues on the BA.
  • L 2 is a linker which binds to the BA through a cysteine residue.
  • the subscript, t represents the number of linkers, L 2 , bonded to the BA through cysteine residues on the BA.
  • t is an integer from 0 to 8.
  • the linker, L 2 when the linker, L 2 , is a bidentate linker, t is an integer from 0 to 4. In some of these examples, the sum of k + t is equal to 1-8.
  • the compound of Formula I is: O N 3 8 wher , , .
  • L 1 is a linker which binds to the BA through a lysine residue.
  • L 2 is a linker which binds to the BA through a cysteine residue.
  • the compound of Formula I is:
  • L 1 is a linker which binds to the BA through a lysine residue.
  • L 2 is a linker which binds to the BA through a cysteine residue.
  • R 1 is, independently at each occurrence, halo, haloalkyl, haloalkoxy, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, aryl, alkaryl, aralkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitro id
  • R A is alkyl o
  • q is an integer from 0 to 5.
  • the linker is:
  • AA is an amino acid
  • the spacer is a divalent moiety that connects the AA 1 -AA 2 moiety to the binding agent (BA).
  • Suitable spacers include, but are not limited to, those comprising alkylene or polyethylene glycol.
  • the ends of the spacers, i.e., the portion of the spacer directly bonded to the binding agent or AA 1 can be moieties derived from reactive moieties that are used for purposes of coupling the antibody or AA 1 to the spacer during the chemical synthesis of the conjugate.
  • the spacer comprises an alkylene. In some embodiments, the spacer comprises a C 5-7 alkylene. In some embodiments, the spacer is:
  • the spacer is:
  • the spacer is:
  • R N is a hydrogen atom or alkyl
  • R M is alkyl; the two bonds represented b e bonds to cysteines of a binding agent; and
  • b is an integer from 2 to 8
  • the spacer is: h i
  • the spacer is:
  • s an integer from 2 to 20. In some embodiments, g is 2-8. In some embodiments, g is 2, 4, 6, or 8.
  • the spacer is:
  • AA 1 -AA 2 is: valine-citrulline, citrulline-valine, lysine- phenylalanine, phenylalanine-lysine, valine-asparagine, asparagine-valine, threonine- asparagine, asparagine-threonine, serine-asparagine, asparagine-serine, phenylalanine- asparagine, asparagine-phenylalanine, leucine-asparagine, asparagine-leucine, isoleucine- asparagine, asparagine-isoleucine, glycine-asparagine, asparagine-glycine, glutamic acid- asparagine, asparagine-glutamic acid, citrulline-asparagine, asparagine-citrulline, alanine- asparagine, or asparagine-alanine.
  • AA 1 -AA 2 is: valine-citrulline or citrulline-valine. In some embodiments, AA 1 -AA 2 is: valine-citrulline.
  • the compound of Formula I is:
  • , , , A is aryl or heteroaryl
  • t is an integer from 1-8.
  • the compound of Formula I is:
  • Ab is an antibody
  • t is an integer from 1 to 8. In some examples, k is an integer from 1 to 8. In some examples, t is an integer from 1 to 4. In some examples, when is a bond to a cysteine of the antibody, up to 8 conjugates set forth herein may be bonded to the antibody. In some examples, when is a bond to a lysine of the antibody, up to 30 conjugates set forth herein may be bonded to the antibody.
  • the compound of Formula I is:
  • n some emo men s an neger rom o .
  • n some emo men s an integer from 1 to 8.
  • k is an integer from 1 to 6.
  • k is an integer from 1 to 4.
  • k is an integer from 1 to 3.
  • the drug-antibody ratio (DAR) of the conjugate is from 1.0 to 3.0.
  • these compounds represent the payload portion of the conjugates described herein and are released, e.g., by enzyme proteolysis, following internalization of the conjugate into a cell.
  • the methods provided herein include methods of treating a proliferative disease, e.g., cancer, comprising administering to a patient a therapeutically effective amount of a conjugate, e.g., antibody-drug conjugate that releases a compound of Formula (II) following internalization of said conjugate into a cell in said patient.
  • these compounds represent the metabolic product of the conjugates described herein, e.g., enzyme proteolysis product. In some embodiments, these compounds represent the catabolic product of the conjugates described herein. In some embodiments, these compounds represent the cellular product of the conjugates described herein.
  • A is a divalent radical of benzene, of pyridine, of naphthalene, or of quinolone, which are optionally substituted.
  • A is arylene
  • A is:
  • n is an integer from 0 to 4.
  • n is and integer from 0 to 3;
  • p is an integer from 0 to 6;
  • q is an integer from 0 to 5.
  • the compound of Formula (II) is a compound of the Formula (IIA):
  • R 1 and n are as defined herein.
  • the compound of Formula (II) is a compound of the Formula (IIB):
  • R 1 and q are as defined herein.
  • the compound of Formula (II) is a compound of the Formula (IIB2):
  • R 1 and q are as defined herein.
  • the compound of Formula (II) is a compound of the Formula (IIB3):
  • R 1 and q are as defined herein.
  • the compound of Formula (II) is a compound of the Formula (IIC):
  • R 1 and q are as defined herein.
  • the compound of Formula (II) is a compound of the Formula (IID):
  • R 1 and q are as defined herein.
  • the compound of Formula (II) is a compound of the Formula (IIE):
  • R 1 and q are as defined herein.
  • the compound of Formula (II) is a compound of the Formula (IIF):
  • the compound of Formula (II) is a compound of the Formula (IIG):
  • R 1 and q are as defined herein.
  • R 1 is, independently, alkyl or halo. In some embodiments, R 1 is, independently, C 1-6 alkyl, C 1-6 haloalkyl, or halo. In some embodiments, R 1 is, independently, C 1-6 haloalkyl or halo. In some embodiments, R 1 is, independently, halo. In some embodiments, R 1 is, independently, fluoro, chloro, bromo, iodo, or m, p, or q is 0 or 1. In some embodiments, n, m, p, or q is 0.
  • R 1 is, independently, alkyl, alkoxy, heteroalkyl, halo, haloalkyl, or haloalkoxy. In some embodiments, R 1 is, independently, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 haloalkoxy, or halo. In some embodiments, R 1 is, independently, C 1-6 alkyl or C 1-6 alkoxy. In some embodiments, R 1 is, independently, alkoxy. In some embodiments, R 1 is, independently, methoxy, ethoxy, propoxy. In some embodiments, n, m, p, or q is 0, 1 or 2.
  • n, m, p, or q is 0 or 1. In some embodiments, n, m, p, or q is 0.In some embodiments, the compound of Formula (II) is a compound of Formula (IIA):
  • R 1 is, independently at each occurrence, halo or trifluoromethyl; and n is 0, 1, or 2.
  • the compound of Formula (II) is a compound of Formula (IIB):
  • R 1 is, independently at each occurrence, halo or trifluoromethyl; and q is 0, 1, or 2.
  • the compound of Formula (II) is:
  • the methods provided herein include methods of treating a proliferative disease, e.g., cancer, comprising administering to a patient a therapeutically effective amount of a conjugate, e.g., antibody-drug conjugate that releases a patient.
  • these compounds represent the metabolic product of the conjugates described herein, e.g., enzyme proteolysis product.
  • A is a divalent radical of benzene, of pyridine, of naphthalene, or of quinolone, which are optionally substituted.
  • A is arylene
  • A is:
  • R 1 is, independently at each occurrence, alkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl, halo, haloalkoxy, heteroaryl, heterocycloalkyl, hydroxyl, cyano, nitro, , , , or azido,
  • n is an integer from 0 to 4.
  • n is and integer from 0 to 3;
  • p is an integer from 0 to 6;
  • q is an integer from 0 to 5.
  • the compound of Formula (II) is a compound of the Formula (IIA):
  • R 1 is, independently at each occurrence, methoxy, halo or trifluoromethyl; and n is 0, 1, or 2.
  • the compound of Formula (II) is a compound of the Formula (IIB2):
  • the compound of Formula (II) is a compound of the Formula (IIB3):
  • R 1 is, independently at each occurrence, methoxy, halo or trifluoromethyl; and q is 0, 1, or 2.
  • R 1 is, independently, alkyl or halo.
  • R 1 is, independently, C 1-6 alkyl, C 1-6 haloalkyl, or halo.
  • R 1 is, independently, C 1-6 haloalkyl or halo.
  • R 1 is, independently, halo.
  • R 1 is, independently, fluoro, chloro, bromo, iodo, or trifluoromethyl.
  • n, m, p, or q is 0, 1 or 2.
  • n, m, p, or q is 0 or 1.
  • n, m, p, or q is 0.
  • R 1 is, independently, alkyl, alkoxy, heteroalkyl, halo, haloalkyl, or haloalkoxy. In some embodiments, R 1 is, independently, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, C 1-6 haloalkoxy, or halo. In some embodiments, R 1 is, independently, C 1-6 alkyl or C 1-6 alkoxy. In some embodiments, R 1 is, independently, alkoxy. In some embodiments, R 1 is, independently, methoxy, ethoxy, propoxy. In some embodiments, n, m, p, or q is 0, 1 or 2. In some embodiments, n, m, p, or q is 0 or 1. In some embodiments, n, m, p, or q is 0.
  • the compound of Formula (II) is:
  • R 1 and n are as defined herein.
  • the compound of Formula (II) is a compound selected from C H 3 CH3 CH
  • Compounds of Formula I can be synthesized by coupling compounds of Formula P1 with a binding agent, e.g., antibody under standard conjugation conditions (see, e.g, Doronina et al., Nature Biotechnology 2003, 21, 7, 778, which is incorporated herein by reference).
  • a binding agent e.g., antibody under standard conjugation conditions
  • the binding agent is an antibody
  • the antibody can be coupled to a compound of Formula P1 via one or more cysteine or lysine residues of the antibody.
  • Compounds of Formula P1 can be coupled to cysteine residues, for example, by subjecting the antibody to a reducing agent, e.g., dithiotheritol, to cleave the disulfide bonds of the antibody, purifying the reduced antibody, e.g., by gel filtration, and subsequently reacting the antibody with a compound of formula P1 containing a reactive moiety, e.g., a maleimido group.
  • Suitable solvents include, but are not limited to water, DMA, DMF, and DMSO.
  • Compounds of formula P1 containing a reactive moiety, e.g., activated ester or acid halide group can be coupled to lysine residues.
  • Suitable solvents include, but are not limited to protein techniques, including, for example, size exclusion chromatography, dialysis, and ultrafiltration/diafiltration.
  • RL is a reactive linker
  • A is arylene or heteroarylene
  • L is a linker
  • BA is a binding agent
  • the compound of formula P1 includes A, wherein A is:
  • the reactive linker is a moiety comprising a portion in its structure that is capable of reacting with the binding agent (e.g., reacting with an antibody at its cysteine or lysine residues) to form the compound of Formula I. Following conjugation to the binding agent, the reactive linker becomes the linker (L) moiety of the compound of Formula I.
  • Illustrative reactive linkers include, but are not limited to, those that comprise haloacetyl, isothiocyanate, or maleimide portions that are capable of reacting with the binding agent. Reactive portions also include moieties having the following structure:
  • the reactive linker is: wherein:
  • SP R is a reactive spacer
  • AA 1 is an amino acid
  • AA 2 is an amino acid.
  • the reactive spacer is a moiety that contains the above-described reactive linker portion that is capable of reacting with the binding agent and connects this portion to AA 1 .
  • Suitable spacers include, but are not limited to, those comprising alkylene or polyethylene glycol connecting the AA 1 to the portion capable of reacting with binding agent (e.g., haloacetyl, isothiocyanate, or maleimide).
  • the reactive spacer comprises a non-cleavable moiety selected from
  • n 4, 5, 6, 7, 8, 9, or 10.
  • the reactive spacer is
  • the reactive spacer is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • the reactive spacer is:
  • the reactive spacer is: wherein b is an
  • the reactive spacer is:
  • aline lysine- phenylalanine, phenylalanine-lysine, valine-asparagine, asparagine-valine, threonine- asparagine, asparagine-threonine, serine-asparagine, asparagine-serine, phenylalanine- asparagine, asparagine-phenylalanine, leucine-asparagine, asparagine-leucine, isoleucine- asparagine, asparagine-isoleucine, glycine-asparagine, asparagine-glycine, glutamic acid- asparagine, asparagine-glutamic acid, citrulline-asparagine, asparagine-citrulline, alanine- asparagine, or asparagine-alanine.
  • AA 1 -AA 2 is: valine-citrulline or citrulline-valine. In some embodiments, AA 1 -AA 2 is: valine-citrulline.
  • the reactive linker is:
  • SP R is a reactive spacer
  • R AA1 is an amino acid side chain
  • R AA2 is an amino acid side chain.
  • the reactive linker is:
  • SP is a reactive spacer
  • the reactive linker is:
  • the reactive linker is:
  • the reactive linker is:
  • b is an integer from 2 to 8.
  • the reactive linker is:
  • the reactive linker is: wherein b is an inte , , d R M is alkyl. [0323] In some embodiments, the reactive linker is:
  • the reactive linker is:
  • the reactive linker is:
  • R M is alkyl
  • the reactive linker is: . wherein b is an integ
  • the reactive linker is:
  • the reactive linker is:
  • the reactive linker is:
  • the reactive linker is: [0331] In some embodiments, the reactive linker is:
  • the reactive linker is:
  • the compound of Formula P1 is a compound of Formula P1A:
  • A is:
  • R is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heterocycloalkyl, hydroxyl, cyano, nitr , or azido, wherein R A is alkyl
  • n is an integer rom 0 to 4.
  • n is an integer from 0 to 3;
  • p is an integer from 0 to 6;
  • q is an integer from 0 to 5;
  • SP R is a reactive spacer
  • AA 1 is an amino acid
  • AA 2 is an amino acid.
  • the compound of Formula P1A is a compound which includes A wherein A is:
  • the compound of Formula P1A is a compound which includes A wherein A is:
  • n is 0 or 1; and R 1 is C 1-6 alko 1-6 haloalkyl.
  • the compound of Formula P1A is a compound which includes A wherein A is:
  • q is an integer from 0 to 5; and alkoxy, halo, or C 1-6 haloalkyl.
  • the compound of Formula P1A is a compound which includes A wherein A is:
  • q is an integer from 0 to 5; and alkoxy, halo, or C 1-6 haloalkyl.
  • the compound of Formula P1A is a compound which includes A wherein A is:
  • n is 0 or 1; and R 1 is alkoxy, ha alkyl.
  • R 1 is methylsulfonyl, N-methylformamide, hydroxyl, or morpholinyl.
  • the compound of Formula P1 is a compound of Formula P1B:
  • A is:
  • n is an integer from 0 to 3;
  • p is an integer from 0 to 6;
  • q is an integer from 0 to 5;
  • SP R is a reactive spacer
  • the compound of Formula P1 is a compound of Formula P1C:
  • AA 1 is an amino acid
  • AA 2 is an amino acid
  • R 1 is, independently at each occurrence, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl, and
  • n 0, 1, or 2.
  • the compound of Formula P1 is a compound of Formula P1D:
  • SP R is a reactive spacer
  • AA 1 is an amino acid
  • AA 2 is an amino acid
  • R 1 is, independently at each occurrence, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl, and
  • n 0, 1, or 2. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2.
  • the compound of Formula P1 is a compound of Formula P1D, wherein R 1 is alkoxy, halo, or haloalkyl. In some embodiments, R 1 is C 1-6 alkoxy, halo, or C 1-6 haloalkyl. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2.
  • the compound of Formula P1 is a compound of Formula P1D, wherein R 1 is C 1-6 alkoxy, halo, or C 1-6 haloalkyl; and SP R -AA 1 -AA 2 is , wherein b is an integer from 2 to 8 and is a bodiments, n is 0. In some embodiments, n is 1. In
  • n is 2. In some embodiments b is 2. In some embodiments b is 3. In some embodiments b is 4. In some embodiments b is 5. In some embodiments b is 6. In some embodiments b is 7. In some embodiments b is 8.
  • the compound of Formula P1 is a compound of Formula P1E:
  • SP R is a reactive spacer
  • R AA1 is an amino acid side chain
  • R AA2 is an amino acid side chain
  • R 1 is, independently at each occurrence, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl, and
  • n 0, 1, or 2.
  • the compound of Formula P1 is a compound of Formula P1F:
  • SP R is a reactive spacer
  • R 1 is, independently at each occurrence, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl, and
  • n 0, 1, or 2.
  • the compound of Formula P1 is a compound of Formula P1G:

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Abstract

Provided herein are maytansinoid derivatives, conjugates thereof, and methods of treating or preventing proliferative diseases with the same.

Description

INTERNATIONAL PCT PATENT APPLICATION MAYTANSINOID DERIVATIVES,
CONJUGATES THEREOF, AND METHODS OF USE
[0001] This application claims priority to, and the benefit of, US Provisional Patent Application No.62/139,044, entitled MAYTANSINOID DERIVATIVES, CONJUGATES THEREOF AND METHODS OF TREATING PROLIFERATIVE DISEASES USING THE SAME, which was filed March 27, 2015, and also claims priority to, and the benefit of US Provisional Patent Application No.62/252,239, entitled MAYTANSINOID DERIVATIVES, CONJUGATES THEREOF AND METHODS OF TREATING PROLIFERATIVE DISEASES USING THE SAME, which was filed Novemer 6, 2015. The contents of each of these provisional patent applications are herein incorporated by reference in their entirety for all purposes. FIELD [0002] The present disclosure concerns maytansinoid derivatives, conjugates thereof, and methods of treating or preventing proliferative diseases with the same. BACKGROUND [0003] Proliferative diseases, for example cancer, are characterized by the uncontrolled growth of abnormal cells. Current treatments of proliferative diseases include surgery, radiation, chemotherapy, hormone-based therapy and/or immunotherapy. A number of these treatments, particularly chemotherapy, utilize anti-proliferative drugs that limit the spread of the abnormal cells. However, these drugs are typically indiscriminate in their ability to kill cells, affecting both normal and abnormal cells. To address this problem, various approaches to targeted drug delivery have been explored, including the use of conjugates of tumor-targeted probes (such as antibodies or growth factors) with toxins, to selectively target abnormal cells. Antibody drug conjugates (ADCs) are compounds composed of an antibody that is linked, via a chemical linker, to a cytotoxic agent. Such compounds leverage the antibody’s binding specificity for its target to deliver a cytotoxic agent to an abnormal cell. Thus, there is a need for anti-proliferative compounds and their conjugates. [0004] Provided herein are compounds of Formula (I):
Figure imgf000004_0001
or a pharmaceutically acceptable salt thereof,
wherein:
A is arylene or heteroarylene;
L is a linker;
BA is a binding agent; and
k is an integer from 1 to 30. Also provided herein are stereoisomers of compounds of Formula (I).
[0005] Provided herein are also compounds of Formula (II):
Figure imgf000004_0002
or a pharmaceutically acceptable salt thereof, wherein A is arylene or heteroarylene. Also provided herein are stereoisomers of compounds of Formula (II).
[0006] Provided herein are also compounds of Formula PP5:
Figure imgf000005_0001
or a salt thereof, wherein A is arylene or heteroarylene. Also provided herein are stereoisomers of compounds of Formula PP5.
[0007] Provided herein are also compounds of Formula PT1:
Figure imgf000005_0002
PT1 or a salt thereof, wherein A is arylene or heteroarylene and L is a linker. Also provided herein are stereoisomers of compounds of Formula PT1.
[0008] Furthermore, provided herein are methods of treating proliferative diseases comprising administering the compounds described herein. [0009] Furthermore, provided herein are methods of treating proliferative diseases comprising administering the conjugates described herein. [0010] Furthermore, provided herein are of methods of preparing compounds of Formula (I) comprising reacting a deglycosylated antibody or aglycosylated antibody with a compound of Formula (PT1) in the presence of transglutaminase. BRIEF DESCRIPTIONS OF THE DRAWINGS alanine-4-aminobenzamido-citrulline-valine-caprolyl-6-maleimidyl.
[0012] FIG 2 depicts the plot of % Cell Viability vs. Log10 [M] of certain compounds tested in EXAMPLE 41.
[0013] FIG 3 depicts the plot of % Cell Viability vs. Log10 [M] of certain compounds tested in EXAMPLE 41.
[0014] FIG.4 depicts the plot of % Cell Viability vs. Log10 [M] of certain compounds tested in EXAMPLE 41.
[0015] FIG.5 depicts the plot of % Cell Viability vs. Log10 [M] of certain compounds tested in EXAMPLE 41.
[0016] FIG.6 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(4-amino-2-fluoro)benzamido-Cit-Val-Cap-Mal.
[0017] FIG.7 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(4-amino-2-trifluoromethyl)benzamido-Cit-Val-Cap-Mal.
[0018] FIG.8 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(4-amino-2-methoxy)benzamido-Cit-Val-Cap-Mal.
[0019] FIG.9 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-4-aminobenzamide.
[0020] FIG.10 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(2-fluoro-4-amino)benzamide
[0021] FIG.11 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(2-trifluoromethyl-4-amino)benzamide.
[0022] FIG.12 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(2-methoxy-4-amino)benzamide.
[0023] FIG.13 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(3-trifluoromethyl-4-amino)benzamide.
[0024] FIG.14 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(2-chloro-4-amino-5-fluoro)benzamide.
[0025] FIG.15 depicts a general synthetic sequence for preparing compounds of Formula (II) wherein substituent R is defined herein and below.
[0026] FIG.16 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(2,5-difluoro-4-amino)benzamide.
[0027] FIG.17 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(3-fluoro-4-amino)benzamide alanine-(3-chloro-4-amino)benzamide.
[0029] FIG.19 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(5-amino-8-carboxyquinoline)carboxamide.
[0030] FIG.20 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(3-bromo-4-amino)benzamide.
[0031] FIG.21 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(3-methoxy-4-amino)benzamide.
[0032] FIG.22 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(2-methyl-4-amino)benzamide.
[0033] FIG.23 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(3-methyl-4-amino)benzamide.
[0034] FIG.24 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(8-amino-5-carboxyquinoline)carboxamide.
[0035] FIG.25 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(3-methoxy-4-amino)benzamido-Cit-Val-Cap-Mal.
[0036] FIG.26 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-(2-fluoro-4-amino)benzamido-Cit-Val-Cap-6-amine.
[0037] FIG.27 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(2-methoxy-5-amino)benzamide.
[0038] FIG.28 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(3-amino-4-methoxy)benzamide.
[0039] FIG.29 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(3-amino-5-fluoro)benzamide.
[0040] FIG.30 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(2-fluoro-5-amino)benzamide.
[0041] FIG.31 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(3-amino)benzamide.
[0042] FIG.32 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(3-amino-4-fluoro)benzamide.
[0043] FIG.33 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-4-aminobenzamide-adipic-NHS.
[0044] FIG.34 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-4-aminobenzamide-Cap-Mal. alanine-N-(3-methylsulfonyl-4-amino)benzamide.
[0046] FIG.36 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(3-hydroxy-4-amino)benzamide.
[0047] FIG.37 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(2-amino)benzamide.
[0048] FIG.38 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(4-methoxy-2-amino)benzamide.
[0049] FIG.39 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(3-morpholino-4-amino)benzamide.
[0050] FIG.40 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-(3-acetamido-4-amino)benzamide.
[0051] FIG.41 depicts a synthetic sequence for preparing maytansin-N-methyl-L- alanine-N-4-aminobenzamide-Cit-Val-cap-diBromomethylacryl.
[0052] FIG.42 depicts the deconvoluted mass spectroscopy (MS) spectrum of the antibody drug conjugate, PRLR-Q-63 conjugate from EXAMPLE 43.
[0053] FIG.43 depicts the deconvoluted MS spectrum of the Isotype Control-Q-63 conjugate from EXAMPLE 43.
[0054] FIG.44 depicts the plot of % Cell Viability vs. Log10 [M] of certain compounds tested in EXAMPLE 45.
[0055] FIG.45 depicts the plot of % Cell Viability vs. Log10 [M] of certain compounds tested in EXAMPLE 45.
[0056] FIG.46 depicts the plot of % Cell Viability vs. Log10 [M] of certain compounds tested in EXAMPLE 45.
[0057] FIG.47 depicts the plot of % Cell Viability vs. Log10 [M] of certain compounds tested in EXAMPLE 45.
DETAILED DESCRIPTION A. DEFINITIONS [0058] As used herein,“alkyl” refers to a monovalent and saturated hydrocarbon radical moiety. Alkyl is optionally substituted and can be linear, branched, or cyclic, i.e., cycloalkyl. Alkyl includes, but is not limited to, those having 1-20 carbon atoms, i.e., C1-20 alkyl; 1-12 carbon atoms, i.e., C1-12 alkyl; 1-8 carbon atoms, i.e., C1-8 alkyl; 1-6 carbon atoms, are not limited to methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, i-butyl, a pentyl moiety, a hexyl moiety, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
[0059] As used herein,“haloalkyl” refers to alkyl, as defined above, wherein the alkyl includes at least one substituent selected from a halogen, e.g., F, Cl, Br, or I.
[0060] As used herein,“alkenyl” refers to a monovalent hydrocarbon radical moiety containing at least two carbon atoms and one or more non-aromatic carbon-carbon double bonds. Alkenyl is optionally substituted and can be linear, branched, or cyclic. Alkenyl includes, but is not limited to, those having 2-20 carbon atoms, i.e., C2-20 alkenyl; 2-12 carbon atoms, i.e., C2-12 alkenyl; 2-8 carbon atoms, i.e., C2-8 alkenyl; 2-6 carbon atoms, i.e., C2-6 alkenyl; and 2-4 carbon atoms, i.e., C2-4 alkenyl. Examples of alkenyl moieties include, but are not limited to vinyl, propenyl, butenyl, and cyclohexenyl.
[0061] As used herein,“alkynyl” refers to a monovalent hydrocarbon radical moiety containing at least two carbon atoms and one or more carbon-carbon triple bonds. Alkynyl is optionally substituted and can be linear, branched, or cyclic. Alkynyl includes, but is not limited to, those having 2-20 carbon atoms, i.e., C2-20 alkynyl; 2-12 carbon atoms, i.e., C2-12 alkynyl; 2-8 carbon atoms, i.e., C2-8 alkynyl; 2-6 carbon atoms, i.e., C2-6 alkynyl; and 2-4 carbon atoms, i.e., C2-4 alkynyl. Examples of alkynyl moieties include, but are not limited to ethynyl, propynyl, and butynyl.
[0062] As used herein,“alkoxy” refers to a monovalent and saturated hydrocarbon radical moiety wherein the hydrocarbon includes a single bond to an oxygen atom and wherein the radical is localized on the oxygen atom¸e.g.¸CH3CH2-O· for ethoxy. Alkoxy substituents bond to the compound which they substitute through this oxygen atom of the alkoxy substituent. Alkoxy is optionally substituted and can be linear, branched, or cyclic, i.e., cycloalkoxy. Alkoxy includes, but is not limited to, those having 1-20 carbon atoms, i.e., C1-20 alkoxy; 1-12 carbon atoms, i.e., C1-12 alkoxy; 1-8 carbon atoms, i.e., C1-8 alkoxy; 1-6 carbon atoms, i.e., C1-6 alkoxy; and 1-3 carbon atoms, i.e., C1-3 alkoxy. Examples of alkoxy moieties include, but are not limited to methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s- butoxy, t-butoxy, i-butoxy, a pentoxy moiety, a hexoxy moiety, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy.
[0063] As used herein,“haloalkoxy” refers to alkoxy, as defined above, wherein the alkoxy includes at least one substituent selected from a halogen, e.g., F, Cl, Br, or I.
[0064] As used herein,“aryl” refers to a monovalent moiety that is a radical of an aromatic compound wherein the ring atoms are carbon atoms. Aryl is optionally substituted include, but are not limited to those having 6 to 20 ring carbon atoms, i.e., C6-20 aryl; 6 to 15 ring carbon atoms, i.e., C6-15 aryl, and 6 to 10 ring carbon atoms, i.e., C6-10 aryl. Examples of aryl moieties include, but are limited to phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, and pyrenyl.
[0065] As used herein,“arylene” refers to a divalent moiety of an aromatic compound wherein the ring atoms are only carbon atoms. Arylene is optionally substituted and can be monocyclic or polycyclic, e.g., bicyclic or tricyclic. Examples of aryl moieties include, but are not limited to those having 6 to 20 ring carbon atoms, i.e., C6-20 arylene; 6 to 15 ring carbon atoms, i.e., C6-15 arylene, and 6 to 10 ring carbon atoms, i.e., C6-10 arylene.
[0066] As used herein,“alkaryl” refers to an aryl that is substituted with at least one alkyl. Alkaryl is optionally substituted.
[0067] As used herein,“heteroalkyl” refers to an alkyl in which one or more carbon atoms are replaced by heteroatoms. As used herein,“heteroalkenyl” refers to an alkenyl in which one or more carbon atoms are replaced by heteroatoms. As used herein,
“heteroalkynyl” refers to an alkenyl in which one or more carbon atoms are replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen, oxygen, and sulfur atoms. Heteroalkyl is optionally substituted. Examples of heteroalkyl moieties include, but are not limited to, aminoalkyl, sulfonylalkyl, sulfinylalkyl. Examples of heteroalkyl moieties also include, but are not limited to, methylamino, methylsulfonyl, and methylsulfinyl.
[0068] As used herein,“heteroaryl” refers to a monovalent moiety that is a radical of an aromatic compound wherein the ring atoms contain carbon atoms and at least one oxygen, sulfur, nitrogen, or phosphorus atom. Examples of heteroaryl moieties include, but are not limited to those having 5 to 20 ring atoms; 5 to 15 ring atoms; and 5 to 10 ring atoms.
Heteroaryl is optionally substituted.
[0069] As used herein,“heteroarylene” refers to an arylene in which one or more ring atoms of the aromatic ring are replaced with an oxygen, sulfur, nitrogen, or phosphorus atom. Heteroarylene is optionally substituted.
[0070] As used herein,“heterocycloalkyl” refers to a cycloalkyl in which one or more carbon atoms are replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen, oxygen, and sulfur atoms. Heterocycloalkyl is optionally substituted. Examples of heterocycloalkyl moieties include, but are not limited to, morpholinyl, piperidinyl, dithiolanyl, oxanyl, or thianyl.
[0071] As used herein,“optionally substituted,” when used to describe a radical moiety, e.g., optionally substituted alkyl, means that such moiety is optionally bonded to one or more substituents. Examples of such substituents include, but are not limited to halo, cyano, nitro, haloalkyl, azido, epoxy, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, , , , , , , , , , , or , wherein RA, RB, and RC are, independently at each occurrence, a hydrogen atom, alkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl, heteroalkyl, heteroaryl, or heterocycloalkyl, or RA and RB, together with the atoms to which they are bonded, form a saturated or unsaturated carbocyclic ring, wherein the ring is optionally substituted and wherein one or more ring atoms is optionally replaced with a heteroatom. In some embodiments, RA, RB, and RC are not hydrogen atoms. In some examples, RA is methyl. In some examples, RA is
methylamino, methylsulfonyl, and methylsulfinyl. In some examples, RA is methylamino. In certain embodiments, when a radical moiety is optionally substituted with an optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted saturated or unsaturated carbocyclic ring, the substituents on the optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted saturated or unsaturated carbocyclic ring, if they are substituted, are not substituted with substituents which are further optionally substituted with additional substituents. In some embodiments, when a group described herein is optionally substituted, the substituent bonded to the group is unsubstituted unless otherwise specified.
[0072] As used herein,“binding agent” refers to any molecule capable of binding with specificity to a given binding partner.
[0073] As used herein,“linker” refers to a divalent moiety that covalently links the binding agent to the maytansinoid derivatives described herein.
[0074] As used herein,“amide synthesis conditions” refers to reaction conditions suitable to effect the formation of an amide, e.g., by the reaction of a carboxylic acid, activated carboxylic acid, or acyl halide with an amine. In some examples, amide synthesis conditions refers to reaction conditions suitable to effect the formation of an amide bond first converted to an activated carboxylic acid before the activated carboxylic acid reacts with an amine to form an amide. Suitable conditions to effect the formation of an amide include, but are not limited to, those utilizing reagents to effect the reaction between a carboxylic acid an amine, including, but not limited to, dicyclohexylcarbodiimide (DCC),
diisopropylcarbodiimide (DIC), (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate (PyBOP), (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP), O-(benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU), O-(benzotriazol-1-yl)- N,N,N’,N’-tetramethyluronium tetrafluoroborate (TBTU), 1- [Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid
hexafluorophosphate (HATU), 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), 1- Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 2-Chloro-1,3- dimethylimidazolidinium hexafluorophosphate (CIP), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), and carbonyldiimidazole (CDI). In some examples, a carboxylic acid is first converted to an activated carboxylic ester before reacting with an amine to form an amide bond. In certain embodiments, the carboxylic acid is reacted with a reagent. The reagent activates the carboxylic acid by deprotonating the carboxylic acid and then forming a product complex with the deprotonated carboxylic acid as a result of nucleophilic attack by the deprotonated carboxylic acid onto the protonated reagent. For certain carboxylic acids, this activated ester is more susceptible subsequently to nucleophilic attack by an amine than the carboxylic acid is before it is converted. This results in amide bond formation. As such, the carboxylic acid is described as activated. Exemplary reagents include DCC and DIC.
[0075] As used herein, "therapeutically effective amount" refers to an amount (of a compound) that is sufficient to provide a therapeutic benefit to a patient in the treatment or management of a disease or disorder, or to delay or minimize one or more symptoms associated with the disease or disorder.
[0076] Certain groups, moieties, substituents, and atoms are depicted with a wiggly line that intersects a bond or bonds to indicate the atom through which the groups, moieties, substituents, atoms are bonded. For example, a phenyl group that is substituted with a propyl group depicted as: has the following structure:
Figure imgf000013_0001
CH3
H3. As used herein, illustrations showing substituents bonded to a cyclic group
Figure imgf000013_0003
atic, heteroaromatic, fused ring, and saturated or unsaturated cycloalkyl or
heterocycloalkyl) through a bond between ring atoms are meant to indicate, unless specified otherwise, that the cyclic group may be substituted with that substituent at any ring position in the cyclic group or on any ring in the fused ring group, according to techniques set forth herein or which are known in the field to which the instant disclosure pertains. For example,
the group, , wherein subscript q is an integer from 0 to 4 and in which the positions
Figure imgf000013_0002
Figure imgf000014_0003
which the positions of substituents other than R1 which are bonded to the cyclic group through a bond between ring atoms are described generically, includes the following groups in which the positions of these substituents other than R1 are described specifically:
Figure imgf000014_0001
Also, for example, the group, , in which the positions of substituents other than R1 which are bonded to the cyclic group through a bond between ring atoms are described generically, includes the following groups in which the positions of these substituents other than R1 are described specifically:
Figure imgf000014_0002
described specifically, the substituent R1 may be bonded to any ring position in the cyclic group or on any ring in the fused ring group which is not occupied by one of these substituents other than R1. The following non-limiting representative illustrations indicate that the cyclic group can be substituted with the indicated substituent at any ring position or ,
Figure imgf000015_0001
B. CONJUGATES [0077] Provided herein are compounds of Formula (I):
Figure imgf000015_0002
or a pharmaceutically acceptable salt thereof,
wherein:
A is arylene or heteroarylene;
L is a linker;
BA is a binding agent; and
k is an integer from 1 to 30. 1.“A” moieties [0078] In some embodiments, A is arylene. In some embodiments, A is
heteroarylene. In some embodiments, the arylene or heteroarylene is substituted with one or more electron withdrawing groups and/or one or more electron donating groups.
[0079] In some embodiments, A is a divalent radical of benzene, of pyridine, of naphthalene, or of quinolone, which are optionally substituted. substituted with a member selected from the group consisting of amino, amido, alkyl, halo, haloalkyl, alkoxy, and haloalkoxy.
[0081] In some embodiments, A is:
,
wherein:
Figure imgf000016_0001
R1, independently at each occurrence, is alkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl, halo, heteroaryl, heterocycloalkyl, hydroxyl, cyano, nitro, , , , or azido,
Figure imgf000016_0002
wherein RA is alkyl or heteroalkyl;
n is an integer from 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5.
[0082] In some embodiments, A is:
,
wherein:
Figure imgf000016_0003
R1 is, independently at each occurrence, halo, haloalkyl, haloalkoxy, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, aryl, alkaryl, aralkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitr , or azido,
wherein RA is alkyl o
Figure imgf000016_0004
n is an integer from 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5.
[0083] In some embodiments, A is: wherein:
Figure imgf000017_0001
R1 is, independently at each occurrence, halo, haloalkyl, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitro , or azido,
whe
Figure imgf000017_0002
kyl;
n is an integer from 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5.
[0084] In some embodiments, R1 is C1-6 alkyl or C1-6 alkoxy. In some of these embodiments, R1 is methyl, ethyl, methoxy, or ethoxy. In some of these embodiments, R1 is methoxy.
[0085] In some embodiments, R1 is, independently, alkyl or halo. In some embodiments, R1 is, independently, C1-6 alkyl, C1-6 haloalkyl, or halo. In some embodiments, R1 is, independently, halo. In some embodiments, R1 is, independently, fluoro, chloro, bromo, iodo, or trifluoromethyl. In some embodiments, n, m, p, or q is 0, 1 or 2. In some embodiments, n, m, p, or q is 0 or 1. In some embodiments, n, m, p, or q is 0.
[0086] In some embodiments, R1 is . In some embodiments, R1 is wherein RA is methyl. In some embodiments, R1 is hydroxyl. In some embodiments, R1 is N- methylformamide. In some embodiments, R1 is morpholinyl.
[0087] In some embodiments, R1 is, independently, alkyl, alkoxy, or halo. In some embodiments, R1 is, independently, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, C1-6 haloalkoxy, or halo. In some embodiments, R1 is, independently, halo. In some embodiments, R1 is, independently, fluoro, chloro, bromo, iodo, or trifluoromethyl. In some embodiments, n, m, p, or q is 0, 1 or 2. In some embodiments, n, m, p, or q is 0 or 1. In some embodiments, n, m, p, or q is 0.
[0088] In some embodiments, A is: .
[0089] In some embodiments, A
Figure imgf000018_0001
.
[0090] In some embodiments, A
Figure imgf000018_0005
,
[0091] In some embodiments, A
Figure imgf000018_0002
wherein n is 0, 1 or 2.
Figure imgf000018_0006
[0092] In some embodiments, A is:
,
wherein n is 0, 1 or 2.
Figure imgf000018_0003
[0093] In some embodiments, A is:
,
wherein n is 0 or 1; and R1 is alkoxy, halo, or haloalkyl.
[0094] In some embodiments, A is:
,
wherein n is 0 or 1; and R1 is alkoxy, ha
Figure imgf000018_0004
alkyl. In some examples, R1 is alkoxy.
[0095] In some embodiments, A is: ,
wherein n is 0 or 1; and R1 is C1-6 alk 1-6 haloalkyl.
Figure imgf000019_0001
[0096] In some embodiments, A s:
,
wherein n is 0 or 1; and R1 is C1-6 alkox
Figure imgf000019_0002
C1-6 haloalkyl. In some examples, R1 is C1-6 alkoxy.
[0097] In some embodiments, A is:
wherein n is 0 or 1; R1 is C1-6 alkox d L is
, wherein b is an integer from 2 to 8 and is a
Figure imgf000019_0003
[0098] In some embodiments, A is:
,
wherein n is 0 or 1; R1 is C1-6 alkoxy, ha
Figure imgf000019_0004
haloalkyl; and L is
, wherein b is an integer from 2 to 8 and is a
Figure imgf000019_0005
[0099] In some embodiments, A is:
,
wherein n is 0, 1, 2, 3, or 4.
Figure imgf000020_0001
[0100] In some embodiments, A is:
,
wherein n is 0, 1, 2, 3, or 4.
Figure imgf000020_0002
[0101] In some embodiments, A is:
wherein:
R1 is C
Figure imgf000020_0005
1-6 alkyl, halo, or C1-6 haloalkyl; and
n is 0, 1 or 2.
[0102] In some embodiments, A is:
,
[0103] wherein:
Figure imgf000020_0003
[0104] R1 is C1-6 alkyl, halo, or C1-6 haloalkyl; and
[0105] n is 0, 1 or 2.
[0106] In some embodiments, A is:
wherein:
R1 is C1-6 alkyl, C1-6 l, or C1-6 haloalkoxy; and
Figure imgf000020_0004
n is 0, 1, 2, 3, or 4.
[0107] In some embodiments, A is: ,
wherein:
Figure imgf000021_0001
R1 is C1-6 alkyl, C1-6 alkoxy, halo, C1-6 haloalkyl, or C1-6 haloalkoxy; and n is 0, 1, 2, 3, or 4.
[0108] In some embodiments, A is:
,
wherein R1 is C1-6 alkyl, C1-6 alkoxy,
Figure imgf000021_0002
aloalkyl. In certain of these embodiments, R1 is methoxy or methyl. In some specific embodiments, R1 is methoxy.
[0109] In some embodiments, A is: .
Figure imgf000021_0003
[0110] In some embodiments, A is:
wherein:
Figure imgf000021_0006
R1 is halo or trifluoromethyl; and
n is 0, 1 or 2.
[0111] In some embodiments, A is: .
[0112] In some embodiments,
Figure imgf000021_0004
wherein:
Figure imgf000021_0005
X is a hydrogen atom, halo, or trifluoromethyl.
[0113] In some embodiments, A is: wherein:
Figure imgf000022_0001
X is a hydrogen atom, halo, or trifluoromethyl; is the bond to the nitrogen atom; and is the bond to the carbonyl.
[0114] n some embodiments, A is:
Figure imgf000022_0005
wherein:
Figure imgf000022_0002
R1 is, independently at each occurrence, a hydrogen atom, alkyl, alkoxy, aryl, heteroalkyl, halo, haloalkyl, haloalkoxy or hydroxyl; is the bond to the nitrogen atom; and is the bond to the carbonyl. In some embodiments, R1 is 1-methylethyl- iol, phenyl, 2-fluorophenyl, pyridinyl, 4-pyridinyl, pyrrolidinyl, or 1-
Figure imgf000022_0006
pyrrolidinyl. In some embodiments, R1 is trifluoromethyl. In some embodiments, R1 is methoxy. In some embodiments, R1 is fluoro. In some embodiments, R1 is hydrogen. In some embodiments, A is:
wherein:
Figure imgf000022_0003
R1 is, independently at each occurrence, a hydrogen atom, alkyl, alkoxy, aryl, heteroalkyl, halo, haloalkyl, haloalkoxy, or hydroxyl; is the bond to the nitrogen atom; and is the bond to the carbonyl. In some embodiments, R1 is 1-methylethyl- thiol, phen
Figure imgf000022_0004
yl, luorophenyl, pyridinyl, 4-pyridinyl, pyrrolidinyl, or 1-pyrrolidinyl. In some embodiments, R1 is trifluoromethyl. In some embodiments, R1 is methoxy. In some embodiments, R1 is fluoro. In some embodiments, R1 is hydrogen. methylformamide. In some embodiments, R1 is hydroxyl. In some embodiments, R1 is morpholinyl.
[0116] In some embodiments, A is:
wherein:
R1 is, independently at each occurrence, a hydrogen atom, alkyl, alkoxy, aryl, heteroalkyl, halo, haloalkyl, or haloalkoxy; is the bond to the nitrogen atom; and is the bond to the carbonyl. In some embodiments, R1 is alkyl or alkoxy. In some spec embodiments, R1 is propylamino, difluoro-methoxy, phenyl, 2-fluorophenyl.
Figure imgf000023_0003
In some embodiments, R1 is trifluoromethyl. In some embodiments, R1 is methoxy. In some embodiments, R1 is fluoro. In some embodiments, R1 is hydrogen.
[0117] In some embodiments, A is:
1 2 ; is the bond to the n
Figure imgf000023_0001
and is the bond to the carbonyl. [0118
Figure imgf000023_0004
] In some embodiments, A is:
; is the bond to the n
Figure imgf000023_0002
and is the bond to the carbonyl.
[0119] some embodiments, A is:
Figure imgf000023_0005
; is the bond to the n
Figure imgf000024_0001
and is the bond to the carbonyl.
Figure imgf000024_0005
[0120] In some embodiments, A is:
; is the bond to the n
Figure imgf000024_0002
and is the bond to the carbonyl. [0121]
Figure imgf000024_0006
some embodiments, A is:
, wherein X is F, Cl, Br, CN, methoxy, dimethylamino or cyclopropyl; is the bond to the nitrogen atom; and is the bond to the carbonyl.
[0122] n some embodiments, A is:
Figure imgf000024_0003
, wherein X is F, Cl, Br, CN, methoxy, dimethylamino, 1-methyl-ethyl-thio or
Figure imgf000024_0004
cyclopropyl; is the bond to the nitrogen atom; and is the bond to the carbonyl.
[0123]
Figure imgf000024_0007
n some embodiments, A is: ,
wherein each R1 is independently, at e
Figure imgf000025_0001
ce, a hydrogen atom, alkyl, alkoxy, halo, haloalkyl, or haloalkoxy; is the bond to the nitrogen atom; and is the bond to the carbonyl. In some embodiments, R1 is hydrogen, fluoro, trifl
Figure imgf000025_0005
omethyl, or methoxy. In some embodiments, R1 is fluoro, chloro, bromo, or iodo.
[0124] In some embodiments, A is:
, wherein is the bond to the n
Figure imgf000025_0002
and is the bond to the carbonyl.
[0125] ome embodiments, A is:
Figure imgf000025_0006
,
wherein each R1 is independently, at e
Figure imgf000025_0003
e, a hydrogen atom, alkyl, alkoxy, halo, haloalkyl, or haloalkoxy, is the bond to the nitrogen atom; and is the bond to the carbonyl. [0126]
Figure imgf000025_0007
In some embodiments, A is:
;
Figure imgf000025_0004
is the bond to the carbonyl.
[0127] In some embodiments, A is:
Figure imgf000026_0001
is the bon
Figure imgf000026_0002
d to the nitrogen atom; and is the bond to the carbonyl.
[0128] In some embodiments, A is:
Figure imgf000026_0003
is the bond to the n
Figure imgf000026_0004
t oge ato ; a d is the bond to the carbonyl.
Figure imgf000026_0005
[0129] In some embodiments, A is:
is the bond
Figure imgf000026_0006
to the nitrogen atom; and is the bond to the carbonyl.
[0130]
Figure imgf000026_0007
n some embodiments, A is:
is the bond to the nitr
Figure imgf000026_0008
is the bond to the carbonyl.
[0131]
Figure imgf000026_0009
some embodiments, A is: ; is the bond to the
Figure imgf000027_0001
and is the bond to the carbonyl.
Figure imgf000027_0007
[0132] In some embodiments, A is:
; is the bond to the n
Figure imgf000027_0002
and is the bond to the carbonyl.
[0133] n some embodiments, A is:
Figure imgf000027_0008
.
[0134] I
Figure imgf000027_0003
.
[0135] In some embodiments,
Figure imgf000027_0004
.
Figure imgf000027_0005
[0136] In some embodiments, A is:
,
wherein:
Figure imgf000027_0006
is the bond to the carbonyl. [0137]
Figure imgf000028_0005
In some embodiments, A is:
, wherein:
Figure imgf000028_0001
is the bond to the nitrogen atom; and is the bond to the carbonyl. [0138] n some embodiments, A is:
Figure imgf000028_0006
, wherein:
Figure imgf000028_0002
is the bond to the nitrogen atom; and is the bond to the carbonyl. [0139]
Figure imgf000028_0007
In some embodiments, A is:
, wherein:
Figure imgf000028_0003
is the bond to the nitrogen atom; and is the bond to the carbonyl. [0140] In some embodiments, A is:
Figure imgf000028_0008
.
Figure imgf000028_0004
,
wherein n is 0, 12, or 3.
Figure imgf000029_0001
[0142] In some embodiments, A is:
Figure imgf000029_0002
X s,nepenenty at eac occurrence, a yrogen atom, a y, a oxy, halo, haloalkoxy, haloalkyl, heteroalkyl. In some embodiments, X is fluoro, chloro, bromo, iodo, dimethylamino, methylamino, methoxy, ethoxy, or trifluoromethyl.
[0146] In some embodiments, A is: 1 1 , X
Figure imgf000030_0001
wherein:
X is, independently at each occurrence, a hydrogen atom, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, heteroalkyl. In some embodiments, X is fluoro, chloro, bromo, iodo, dimethylamino, methylamino, methoxy, ethoxy, trifluoromethyl or methoxy;
1
is the bond to the nitrogen atom; and
2
is the bond to the carbonyl. 2. Linkers [0147] The linker portion of the conjugates described herein is a divalent moiety that covalently links the binding agent to the maytansinoid derivatives described herein. Suitable linkers include those that release the maytansinoid portion in the presence of an enzyme or at a particular pH range or value.
[0148] In some embodiments, the linker comprises an enzyme-cleavable moiety. Illustrative enzyme-cleavable moieties include, but are not limited to, peptide bonds, ester linkages, hydrazones, and disulfide linkages. In some embodiments, the linker comprises a cathepsin-cleavable linker.
[0149] In some embodiments, the linker comprises a non-cleavable moiety. In some
embodiments, the non-cleavable linker i or a residue thereof. In
Figure imgf000030_0002
some embodiments, the non-cleavab or a residue thereof.
Figure imgf000031_0001
[0150] Suitable linkers also include, but are not limited to, those that are chemically bonded to two cysteine residues of a single binding agent, e.g., antibody. Such linkers can serve to mimic the antibody’s disulfide bonds that are disrupted as a result of the conjugation process.
[0151] In some embodiments, the linker comprises one or more amino acids. Suitable amino acids include natural, non-natural, standard, non-standard, proteinogenic, non- proteinogenic, and L-, or D- ^ ^ amino acids. In some embodiments, the linker comprises alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, or derivative thereof.
[0152] In some embodiments, the linker comprises valine and citrulline.
[0153] In some embodiments, the linker is:
A SP AA1 AA2
wherein:
SP is a spacer;
Figure imgf000031_0002
A
is one or more bonds to the binding agent;
A1 is an amino acid; and
Figure imgf000031_0003
AA2 is an amino acid.
[0154] The spacer is a divalent moiety that connects the AA1-AA2 moiety to the binding agent (BA). Suitable spacers include, but are not limited to, those comprising alkylene or polyethylene glycol. The ends of the spacers, i.e., the portion of the spacer directly bonded to the binding agent or AA1, can be moieties derived from reactive moieties that are used for purposes of coupling the naked antibody or AA1 to the spacer during the chemical synthesis of the conjugate.
[0155] In some examples, suitable spacers include, but are not limited to, a primary amine-terminated alkylene or a primary amine-terminated polyethylene glycol. The primary amine-terminating end of the spacer can be directly bonded to a deglycosylated antibody or aglycosylated antibody in the presence of transglutaminase. embodiments, the spacer comprises a C5-7 alkylene. In some embodiments, the spacer is:
wherein:
Figure imgf000032_0003
is a bond to the binding agent; and
Figure imgf000032_0004
b is an integer from 2 to 8.
[0157] In some embodiments, the spacer comprises a primary amine-terminated alkylene. In some embodiments, the spacer comprises a NH2-C5-7 alkylene. In some embodiments, the spacer is:
wherein: is a bond to the binding agent; and
s an integer from 2 to 8.
Figure imgf000032_0005
[0158] In some embodiments, the spacer is:
. wh
Figure imgf000032_0001
is a bond to the binding agent.
[0159] In some embodiments, the spacer is:
.
wherein:
Figure imgf000032_0002
[0160]
Figure imgf000033_0007
wh
Figure imgf000033_0001
RN is a hydrogen atom or alkyl;
RM is alkyl; the two bonds represented by are bonds to cysteines of a binding agent; and
b is an integer from 2 to 8.
Figure imgf000033_0006
[0161] In some embodiments, the spacer is:
wherein:
Figure imgf000033_0002
is a bond to the binding agent; and
2 to 8.
Figure imgf000033_0003
[0162] In some embodiments, the spacer is:
wherein:
Figure imgf000033_0004
is a bond to the binding agent; and
ments, g is 2-8. In some
Figure imgf000033_0008
embodiments, g is 2, 4, 6, or 8.
[0163] In some embodiments, the spacer i , wherein n is an integer from 4 to 10. In some embodiments, n is 4, 5, 6,
Figure imgf000033_0005
[0165] In some embodim , wherein n is an integer from 4 to 10. In some embodime
Figure imgf000034_0001
[0166] In some embodiments, the spacer .
[0167] In some embodiments, the spacer
Figure imgf000034_0002
. [0168] In some embodim
Figure imgf000034_0003
en s, e spacer s:
.
[0169] In some embo
Figure imgf000034_0004
.
[0170] In some embo
Figure imgf000034_0005
.
[0171] In some embodime
Figure imgf000034_0006
.
[0172] In some em
Figure imgf000034_0007
.
[0173] In some embo
Figure imgf000035_0001
.
[0174] In some embo
Figure imgf000035_0002
Figure imgf000035_0003
X is N or O; RN and RM are each, independently, hydrogen or alkyl; and b is an integer from 1 to 8.
[0176] In some embodiments, AA1-AA2 is: valine-citrulline, citrulline-valine, lysine- phenylalanine, phenylalanine-lysine, valine-asparagine, asparagine-valine, threonine- asparagine, asparagine-threonine, serine-asparagine, asparagine-serine, phenylalanine- asparagine, asparagine-phenylalanine, leucine-asparagine, asparagine-leucine, isoleucine- asparagine, asparagine-isoleucine, glycine-asparagine, asparagine-glycine, glutamic acid- asparagine, asparagine-glutamic acid, citrulline-asparagine, asparagine-citrulline, alanine- asparagine, or asparagine-alanine.
[0177] In some embodiments, AA1-AA2 is: valine-citrulline or citrulline-valine. In some embodiments, AA1-AA2 is: valine-citrulline.
[0178] In some embodiments, the linker is:
Figure imgf000035_0004
wherein:
SP is a spacer;
Figure imgf000035_0005
substituent bonded to the ^ ^ carbon of an ^-amino acid, including natural and non-natural amino acids. Exemplary amino acid side chains include, but are not limited to, the ^-carbon substituent of alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, and citrulline.
[0180] In some embodiments, the linker is:
Figure imgf000036_0001
[0182] In some embodiments, the linker is:
Figure imgf000036_0002
is a bond to the binding agent; and
is an integer from 2 to 8.
Figure imgf000037_0004
[0183] In some embodiments, BA is an antibody and the linker is:
O NH2
wherein:
Figure imgf000037_0001
the two bonds represented b are bonds to cysteines of the antibody; and b is an integer from 2 to 8.
[0184] In some embodiments, BA i
Figure imgf000037_0005
s an antibody and the linker is:
Figure imgf000037_0002
wherein: the two bonds represented by are bonds to cysteines of the antibody; and b is an integer from 2 to 8
Figure imgf000037_0006
[0185] In some embodiments, BA is an antibody and the linker is:
wherein:
Figure imgf000037_0003
RN is a hydrogen atom or alkyl;
RM is alkyl; b is an integer from 2 to 8.
[0186] In some embodiments, the li
Figure imgf000038_0004
nker is:
wherein:
Figure imgf000038_0001
is a bond to the binding agent; and
is an integer from 2 to 8.
Figure imgf000038_0006
[0187] In some embodiments, the linker is:
Figure imgf000038_0002
wherein: is a bond to the binding agent; and
g is an integer from 2 to 20. In some embodiments, g is 2 to 8. In some
Figure imgf000038_0005
embodiments, g is 2, 4, 6, or 8.
[0188] In some embodiments, the linker is:
.
[0189] In so
Figure imgf000038_0003
.
Figure imgf000039_0001
[0190] In some embodiments, the linker is:
.
[0191] In
Figure imgf000039_0002
O NH2
. [0192] In some em
Figure imgf000039_0003
o mens, e ner s:
.
[0193] In
Figure imgf000039_0004
.
[0194] In s
Figure imgf000040_0001
.
[0195] In s
Figure imgf000040_0002
.
3.
Figure imgf000040_0003
[0196] Suitable binding agents include, but are not limited to, antibodies,
lymphokines, hormones, growth factors, viral receptors, interleukins, or any other cell binding or peptide binding molecules or substances.
[0197] In some embodiments, the binding agent is an antibody. In some
embodiments, the antibody is a monoclonal antibody, polyclonal antibody, antibody fragment (Fab, Fab’, and F(ab)2, minibody, diabody, tribody, and the like), or bispecific antibody. Antibodies herein can be humanized using methods described in US Patent No.6,596,541 and US Publication No.2012/0096572, each incorporated by reference in their entirety.
[0198] Where the binding agent is an antibody, it binds to an antigen binding partner that is a polypeptide and may be a transmembrane molecule (e.g., receptor) or a growth factor that might be glycosylated or phosphorylated. Exemplary antigens include, but are not limited to, molecules such as renin; a growth hormone, including human growth hormone and stimulating hormone; lipoproteins; alpha1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor vmc, factor IX, tissue factor (TF), and von Willebrands factor; anti- clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta;
enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MlP-I-alpha); a serum albumin, such as human serum albumin; Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;
prorelaxin; mouse gonadotropin-associated peptide; a microbial protein, such as
betalactamase; DNase; 19E; a cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; protein A or D; rheumatoid factors; a neurotrophic factor such as bone- derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4, NT-5, or NT- 6), or a nerve growth factor such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; fibroblast growth factor receptor 2 (FGFR2), epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF- β3, TGF-β4, or TGF- β5; insulin-like growth factor-l and -II (IGF-l and IGF-II); des(I-3)-IGF-l (brain IGF-l), insulin-like growth factor binding proteins, EpCAM, GD3, FLT3, PSMA, PSCA, MUC1, MUC16, STEAP, CEA, TENB2, EphA receptors, EphB receptors, folate receptor, FOLRI, mesothelin, cripto, alphavbeta6, integrins, VEGF, VEGFR, EGFR, transferrin receptor, IRTA1, IRTA2, IRTA3, IRTA4, IRTA5; CD proteins such as CD2, CD3, CD4, CD5, CD6, CD8, CD11, CD14, CD19, CD20, CD21, CD22, CD25, CD26, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD59, CD70, CD79, CD80. CD81, CD103, CD105, CD134, CD137, CD138, CD152, or an antibody which binds to one or more tumor-associated antigens or cell-surface receptors disclosed in US Publication No.2008/0171040 or US Publication No.2008/0305044 and incorporated in their entirety by reference; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon, such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; viral antigen such as, for example, a portion of the HIV envelope; transport proteins; homing receptors; addressins; VCAM; a tumor associated antigen such as AFP, ALK, B7H4, BAGE proteins, β-catenin, brc-abl, BRCA1, BORIS, CA9 (carbonic anhydrase IX), caspase-8, CD20, CD40, CD123, CDK4, CEA, CLEC12A, c-kit, cMET, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, endoglin, Epcam, EphA2, ErbB2/Her2, ErbB3/Her3, ErbB4/Her4, ETV6-AML, Fra-1, FOLR1, GAGE proteins (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA/B-raf, HLA/EBNA1, HLA/k-ras, HLA/MAGE-A3, hTERT, IGF1R, LGR5, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML- IAP, Muc1, Muc16 (CA-125), MUM1, NA17, NGEP, NY-BR1, NY-BR62, NY-BR85, NY- ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PDGFR-α, PDGFR-β, PDGF-A, PDGF-B, PDGF-C, PDGF-D, PLAC1, PRLR, PRAME, PSCA, PSGR, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, Steap-1, Steap-2, STn, survivin, TAG- 72, TGF-β, TMPRSS2, Tn, TNFRSF17, TRP-1, TRP-2, tyrosinase, and uroplakin-3, and fragments of any of the above-listed polypeptides.
[0199] Exemplary antigens also include, but are not limited to, BCMA, SLAMF7, B7H4, GPNMB, UPK3A, and LGR5.
[0200] In some embodiments, the antigens include prolactin receptor (PRLR) or prostate-specific membrane antigen (PSMA).
[0201] Binding agents also include, but are not limited to, ankyrin repeat proteins, interferons, lymphokines such as IL-2 or IL-3, hormones like insulin and glucocorticoids, growth factors such as EGF, transferrin and fibronectin type III.
[0202] In some embodiments, the binding agents interact with or bind to tumor antigens, including antigens specific for a type of tumor or antigens that are shared, overexpressed or modified on a particular type of tumor. Examples include, but are not limited to: alpha-actinin-4 with lung cancer, ARTC1 with melanoma, BCR-ABL fusion protein with chronic myeloid leukemia, B-RAF, CLPP or Cdc27 with melanoma, CASP-8 with squamous cell carcinoma, and hsp70-2 with renal cell carcinoma as well as the following shared tumor-specific antigens, for example: BAGE-1, GAGE, GnTV, KK-LC-1, MAGE-A2, NA88-A, TRP2-INT2.
[0203] In some embodiments, the binding agent is an antibody. In some
embodiments, the binding agent is a monoclonal antibody. In some embodiments, the binding agent is a polyclonal antibody. In some embodiments, the antibody is an anti-PSMA, anti-MUC16, or anti-EGFRvIII, or anti-STEAP-2 antibody. binding molecule, through an attachment at a particular amino acid within the antibody or antigen-binding molecule. Exemplary amino acid attachments that can be used in the context of this aspect of the disclosure include, e.g., lysine (see, e.g., US 5,208,020; US
2010/0129314; Hollander et al., Bioconjugate Chem., 2008, 19:358-361; WO 2005/089808; US 5,714,586; US 2013/0101546; and US 2012/0585592), cysteine (see, e.g., US
2007/0258987; WO 2013/055993; WO 2013/055990; WO 2013/053873; WO 2013/053872; WO 2011/130598; US 2013/0101546; and US 7,750,116), selenocysteine (see, e.g., WO 2008/122039; and Hofer et al., Proc. Natl. Acad. Sci., USA, 2008, 105:12451-12456), formyl glycine (see, e.g., Carrico et al., Nat. Chem. Biol., 2007, 3:321-322; Agarwal et al., Proc. Natl. Acad. Sci., USA, 2013, 110:46-51, and Rabuka et al., Nat. Protocols, 2012, 10:1052- 1067), non-natural amino acids (see, e.g., WO 2013/068874, and WO 2012/166559), and acidic amino acids (see, e.g., WO 2012/05982). Linkers can be conjugated via glutamine via transglutaminase-based chemo-enzymatic conjugation (see, e.g., Dennler et al., Bioconjugate Chem.2014, 25, 569-578). Linkers can also be conjugated to an antigen-binding protein via attachment to carbohydrates (see, e.g., US 2008/0305497, WO 2014/065661, and Ryan et al., Food & Agriculture Immunol., 2001, 13:127-130) and disulfide linkers (see, e.g., WO 2013/085925, WO 2010/010324, WO 2011/018611, WO 2014/197854, and Shaunak et al., Nat. Chem. Biol., 2006, 2:312-313).
[0205] In some embodiments, the binding agent is an antibody, and the antibody is bonded to the linker through a lysine residue. In some embodiments, the antibody is bonded to the linker through a cysteine residue.
4. Illustrative embodiments
[0206] In some embodiments,
A is:
,
wherein:
Figure imgf000043_0001
R1, independently at each occurrence, is alkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl, halo, heteroaryl, heterocycloalkyl, hydroxyl, cyano, nitro, , , , or azido,
Figure imgf000043_0002
n is an integer from 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5; and
L is:
wherein:
SP is a spacer;
Figure imgf000044_0002
is one or more bonds to the binding agent;
AA1 is an amino acid; and
Figure imgf000044_0003
AA2 is an amino acid.
[0207] In some embodiments, A is:
,
wherein:
Figure imgf000044_0001
R1, independently at each occurrence, is C1-6 alkyl, C1-6 haloalkyl, or halo; n is an integer from 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5; and
L is:
wherein:
SP is a spacer;
Figure imgf000044_0005
is one or more bonds to the binding agent;
A1 is an amino acid; and
Figure imgf000044_0004
AA2 is an amino acid. , wherein:
Figure imgf000045_0001
R1, independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, haloalkoxy, heteroaryl, heteroalkyl, heterocycloalkyl, hydroxyl, cyano, nitro id
wherein RA is alkyl or heteroalky
n is an integer from 0 to 4;
Figure imgf000045_0003
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5; and
L is:
wherein:
SP is a spacer;
Figure imgf000045_0005
is one or more bonds to the binding agent;
Figure imgf000045_0004
A1 is an amino acid; and AA2 is an amino acid.
[0209] In some embodiments, A is:
, wherein:
Figure imgf000045_0002
R1, independently at each occurrence, is C1-6 alkyl, C1-6 haloalkyl, or halo;
n is an integer from 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5; and wherein:
SP is:
Figure imgf000046_0001
is a bond to the binding agent; and
is an integer from 2 to 8; and
Figure imgf000046_0003
AA1 is an amino acid; and
AA2 is an amino acid.
[0210] In some embodiments, A is:
,
wherein:
Figure imgf000046_0002
R1, independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, hydroxyl, cyano, nitro, , , , or azido,
wherein RA is
n is an intege
Figure imgf000046_0004
r from 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5; and
L is:
wherein:
SP is:
Figure imgf000046_0005
Figure imgf000047_0001
is a bond to the binding agent; and
s an integer from 2 to 8; and
Figure imgf000047_0005
AA1 is an amino acid; and
AA2 is an amino acid.
[0211] In some embodiments, A is:
, wherein:
Figure imgf000047_0002
R1, independently at each occurrence, is C1-6 alkyl, C1-6 haloalkyl, or halo; n is an integer from 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5; and L is:
wherein:
Figure imgf000047_0003
SP is a spacer; is one or more bonds to the binding agent;
Figure imgf000047_0004
, wherein:
Figure imgf000048_0001
R1, independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, hydroxyl, cyan , or azido,
Figure imgf000048_0002
m is an nteger rom 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5; and L is:
wherein:
Figure imgf000048_0003
SP is a spacer;
[ A
Figure imgf000048_0005
, wherein:
Figure imgf000048_0004
R1, independently at each occurrence, is C1-6 alkyl, C1-6 haloalkyl, or halo;
n is an integer from 0 to 4; p is an integer from 0 to 6; and
q is an integer from 0 to 5; and
L is:
wherein:
,
wherein:
Figure imgf000049_0001
R1, independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitro, , , , or azido,
Figure imgf000049_0002
n is an integer from 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5; and
L is:
Figure imgf000049_0003
Figure imgf000050_0001
R1, independently at each occurrence, is C1-6 alkyl, C1-6 haloalkyl, or halo; n is an integer from 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5; and
L is:
wherein:
Figure imgf000050_0002
SP is:
Figure imgf000050_0003
, wherein:
Figure imgf000051_0001
R1, independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, hydroxyl cyano nitro or azido
Figure imgf000051_0004
n is an integer from 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5; and
L is:
wherein:
Figure imgf000051_0002
SP is:
[0217] A is:
Figure imgf000051_0003
, wherein:
Figure imgf000052_0001
R1, independently at each occurrence, is C1-6 alkyl, C1-6 haloalkyl, or halo; and n, m, p, and q are 0, 1, or 2; and L is
Figure imgf000052_0002
g
[0218] In some embodiments, A is:
,
wherein:
Figure imgf000052_0003
R1, independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitro or azido
Figure imgf000052_0004
Figure imgf000053_0001
[0219] In some embodiments,
A is:
;
wherein
Figure imgf000053_0002
R1 is, independently at each occurrence, is C1-6 haloalkyl, or halo; and n is 0, 1, or 2; and
L is:
wherein
[0220]
wherein:
Figure imgf000053_0003
aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, hydroxyl cyano nitro or azido
Figure imgf000054_0001
RAA2 is an amino acid side chain.
[0221] In some embodiments,
A is:
;
wherein
Figure imgf000054_0002
R1 is, independently at each occurrence, is halo; and n is 0, 1, or 2; and
L is:
wherein
Figure imgf000054_0003
wherein:
Figure imgf000055_0001
R1, independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, hydroxyl, cyano, nitr , or azido,
wherein RA is
Figure imgf000055_0002
L is:
Figure imgf000055_0003
wherein n is an integer from 0 to 4; and
b is an integer from 2 to 8.
[0223] In some embodiments,
A is:
Figure imgf000055_0004
Figure imgf000056_0001
wherein:
Figure imgf000056_0002
R1, independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, hyd l it id
Figure imgf000056_0003
.
[0226] In so
Figure imgf000057_0001
wherein:
Figure imgf000057_0002
R1 is, independently at each occurrence, a hydrogen atom, alkyl, alkoxy, aryl, heteroalkyl, halo, haloalkoxy, haloalkyl, or haloalkoxy; is the bond to the nitrogen atom; and is the bond to the carbonyl. In some embodiments, R1 is 1-methylethyl- luorophenyl, pyridinyl, 4-pyridinyl, pyrrolidinyl, or 1-pyrrolidinyl. In some
Figure imgf000057_0004
embodiments, R1 is trifluoromethyl. In some embodiments, R1 is methoxy. In some embodiments, R1 is fluoro. In some embodiments, R1 is hydrogen.
[0227] In some embodiments, A is:
wherein:
Figure imgf000057_0003
R1 is, independently at each occurrence, a hydrogen atom, alkyl, alkoxy, aryl, heteroalkyl, halo, haloalkyl, haloalkoxy; is the bond to the nitrogen atom; and is the bond to the carbonyl. In some embodiments, R1 is 1-methylethyl-
Figure imgf000057_0005
luorophenyl, pyridinyl, 4-pyridinyl, pyrrolidinyl, or 1-pyrrolidinyl. In some embodiments, R1 is trifluoromethyl. In some embodiments, R1 is methoxy. In some embodiments, R1 is fluoro. In some embodiments, R1 is hydrogen.
[0228] In some embodiments,
A is: ; and
L is
Figure imgf000058_0001
.
Figure imgf000058_0002
[0229] In some embodiments,
BA is an antibody,
A is:
wherein
Figure imgf000058_0005
R1 is, independently at each occurrence, is halo; and n is 0, 1, or 2; and
L is:
wherei is a bond
Figure imgf000058_0003
[0230] In some embodiments,
A is:
Figure imgf000058_0006
;
Figure imgf000058_0004
R1, independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitr , or azido; and
q is d
Figure imgf000059_0001
L is:
wherein:
Figure imgf000059_0002
SP is a spacer; is the one or more bonds to the binding agent;
RAA1 is an amino acid side chain; and
RAA2 is an amino acid side chain.
[0231] In some embodiments, A is:
;
wherein:
Figure imgf000059_0003
R1, independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitro, , , , or azido;
Figure imgf000059_0005
q g ;
L is:
wherein:
Figure imgf000059_0004
SP is a spacer; AA1 is an amino acid side chain; and
Figure imgf000060_0005
RAA2 is an amino acid side chain.
[0232] In some embodiments,
A is:
;
wherein:
Figure imgf000060_0001
R1, independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitro , or azido;
wherein q i 5; and
Figure imgf000060_0002
L is:
wherein:
Figure imgf000060_0003
is a bond to the binding agent; and
b is an integer from 2 to 8.
[0233]
Figure imgf000060_0006
In some embodiments,
A is:
;
wherein:
Figure imgf000060_0004
aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitro or azido; and
;
wher
Figure imgf000061_0001
R1, independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitro, , , , or azido;
Figure imgf000061_0002
e
Figure imgf000062_0001
whe
Figure imgf000062_0002
, , , s an integer from 0 to 30, and t is an integer from 0 to 8. In some of these embodiments, L1 is a linker which binds to the BA through a lysine residue. In some of these embodiments, the subscript, k, represents the number of linkers, L1, bonded to the BA through lysine residues on the BA. In some of these embodiments, L2 is a linker which binds to the BA through a cysteine residue. In some of these embodiments, the subscript, t, represents the number of linkers, L2, bonded to the BA through cysteine residues on the BA. In some embodiments, when the linker, L2, is a monodentate linker, t is an integer from 0 to 8. In some
embodiments, when the linker, L2, is a bidentate linker, t is an integer from 0 to 4. In some of these examples, the sum of k + t is equal to 1-8. [0237] In some embodiments, the compound of Formula I is: O N 3 8 wher
Figure imgf000063_0001
, , . In some of these embodiments, L1 is a linker which binds to the BA through a lysine residue. In some of these embodiments, L2 is a linker which binds to the BA through a cysteine residue. [0238] In some embodiments, the compound of Formula I is:
CH H OH OCH 3 3 CH
H OH OCH 3 3
O N O N 3 4 wher
Figure imgf000063_0002
, , . In some of these embodiments, L1 is a linker which binds to the BA through a lysine residue. In some of these embodiments, L2 is a linker which binds to the BA through a cysteine residue. [0239] In some embodiments, A is:
,
wherein:
Figure imgf000063_0003
R1 is, independently at each occurrence, halo, haloalkyl, haloalkoxy, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, aryl, alkaryl, aralkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitro id
wherein RA is alkyl o
n is an integer from 0 to 4;
Figure imgf000063_0004
p is an integer from 0 to 6; and
q is an integer from 0 to 5.
[0240] In some embodiments, the linker is:
w
Figure imgf000064_0003
AA is an amino acid.
[0241] The spacer is a divalent moiety that connects the AA1-AA2 moiety to the binding agent (BA). Suitable spacers include, but are not limited to, those comprising alkylene or polyethylene glycol. The ends of the spacers, i.e., the portion of the spacer directly bonded to the binding agent or AA1, can be moieties derived from reactive moieties that are used for purposes of coupling the antibody or AA1 to the spacer during the chemical synthesis of the conjugate.
[0242] In some embodiments, the spacer comprises an alkylene. In some embodiments, the spacer comprises a C5-7 alkylene. In some embodiments, the spacer is:
Figure imgf000064_0001
[0243] In some embodiments, the spacer is:
.
Figure imgf000064_0002
Figure imgf000065_0005
[0244] In some embodiments, the spacer is:
wh
Figure imgf000065_0001
RN is a hydrogen atom or alkyl;
RM is alkyl; the two bonds represented b e bonds to cysteines of a binding agent; and
b is an integer from 2 to 8
Figure imgf000065_0006
[0245] In some embodiments, the spacer is: h i
Figure imgf000065_0002
is a bond to the binding agent; and
is an integer from 2 to 8.
Figure imgf000065_0007
[0246] In some embodiments, the spacer is:
wherein:
Figure imgf000065_0003
is a bond to the binding agent; and
s an integer from 2 to 20. In some embodiments, g is 2-8. In some
Figure imgf000065_0008
embodiments, g is 2, 4, 6, or 8.
[0247] In some embodiments, the spacer is:
.
Figure imgf000065_0004
. [0249] In some embodi
Figure imgf000066_0001
,
.
[0250] In some embo
Figure imgf000066_0002
.
[0251] In some embo
Figure imgf000066_0003
.
[0252] In some embodimen
Figure imgf000066_0004
.
[0253] In some emb
Figure imgf000066_0005
.
[0254] In some embo
Figure imgf000066_0006
.
[0255] In some embo
Figure imgf000066_0007
Figure imgf000066_0008
Figure imgf000067_0002
1 to 8.
[0257] In some embodiments, AA1-AA2 is: valine-citrulline, citrulline-valine, lysine- phenylalanine, phenylalanine-lysine, valine-asparagine, asparagine-valine, threonine- asparagine, asparagine-threonine, serine-asparagine, asparagine-serine, phenylalanine- asparagine, asparagine-phenylalanine, leucine-asparagine, asparagine-leucine, isoleucine- asparagine, asparagine-isoleucine, glycine-asparagine, asparagine-glycine, glutamic acid- asparagine, asparagine-glutamic acid, citrulline-asparagine, asparagine-citrulline, alanine- asparagine, or asparagine-alanine.
[0258] In some embodiments, AA1-AA2 is: valine-citrulline or citrulline-valine. In some embodiments, AA1-AA2 is: valine-citrulline.
[0259] In some embodiments, the compound of Formula I is:
Figure imgf000067_0001
, , , A is aryl or heteroaryl, and
t is an integer from 1-8.
[0260] In some embodiments, the compound of Formula I is:
Figure imgf000068_0001
,
, , ,
Figure imgf000069_0001
Figure imgf000070_0001
,
,
Figure imgf000071_0001
,
,
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
,
,
Figure imgf000075_0001
H OC 3 3
Figure imgf000076_0001
,
, ,
Figure imgf000077_0001
O N
,
Figure imgf000078_0001
,
, , ,
Figure imgf000079_0001
,
Figure imgf000080_0001
,
,
Figure imgf000081_0001
,
, ,
Figure imgf000082_0001
,
,
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
,
,
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
,
,
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
,
,
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
,
, , ,
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
;
Figure imgf000113_0001
Ab is an antibody;
is a bond to a cysteine of the antibody;
is a bond to a lysine of the antibody;
an integer from 1 to 30; and
Figure imgf000113_0002
t is an integer from 1 to 8. In some examples, k is an integer from 1 to 8. In some examples, t is an integer from 1 to 4. In some examples, when is a bond to a cysteine of the antibody, up to 8 conjugates set forth herein may be bonded to the antibody. In some examples, when is a bond to a lysine of the antibody, up to 30 conjugates set forth herein may be bonded to the antibody.
[0261] In some embodiments, the compound of Formula I is:
Figure imgf000114_0001
Figure imgf000115_0001
n some emo men, s an neger rom o . n some emo men, s an integer from 1 to 8. In some embodiment, k is an integer from 1 to 6. In some embodiments, k is an integer from 1 to 4. In some embodiments, k is an integer from 1 to 3. In some embodiments, the drug-antibody ratio (DAR) of the conjugate is from 1.0 to 3.0. C. MAYTANSINOID DERIVATIVES [0263] Provided herein are compounds of Formula (II):
Figure imgf000115_0002
or a pharmaceutically acceptable salt thereof, [0264] In certain embodiments, these compounds represent the payload portion of the conjugates described herein and are released, e.g., by enzyme proteolysis, following internalization of the conjugate into a cell. The methods provided herein include methods of treating a proliferative disease, e.g., cancer, comprising administering to a patient a therapeutically effective amount of a conjugate, e.g., antibody-drug conjugate that releases a compound of Formula (II) following internalization of said conjugate into a cell in said patient.
[0265] In some embodiments, these compounds represent the metabolic product of the conjugates described herein, e.g., enzyme proteolysis product. In some embodiments, these compounds represent the catabolic product of the conjugates described herein. In some embodiments, these compounds represent the cellular product of the conjugates described herein.
[0266] In some embodiments, A is a divalent radical of benzene, of pyridine, of naphthalene, or of quinolone, which are optionally substituted.
[0267] In some embodiments, A is arylene.
[0268] In some embodiments, A is:
wher
l,
Figure imgf000116_0001
n is an integer from 0 to 4;
m is and integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5.
[0269] In some embodiments, the compound of Formula (II) is a compound of the Formula (IIA):
Figure imgf000117_0001
wherein R1 and n are as defined herein.
[0270] In some embodiments, the compound of Formula (II) is a compound of the Formula (IIB):
Figure imgf000117_0002
wherein R1 and q are as defined herein.
[0271] In some embodiments, the compound of Formula (II) is a compound of the Formula (IIB2):
Figure imgf000117_0003
wherein R1 and q are as defined herein.
[0272] In some embodiments, the compound of Formula (II) is a compound of the Formula (IIB3):
Figure imgf000118_0001
wherein R1 and q are as defined herein.
[0273] In some embodiments, the compound of Formula (II) is a compound of the Formula (IIC):
Figure imgf000118_0002
wherein R1 and q are as defined herein.
[0274] In some embodiments, the compound of Formula (II) is a compound of the Formula (IID):
Figure imgf000118_0003
wherein R1 and q are as defined herein.
[0275] In some embodiments, the compound of Formula (II) is a compound of the Formula (IIE):
Figure imgf000119_0001
wherein R1 and q are as defined herein.
[0276] In some embodiments, the compound of Formula (II) is a compound of the Formula (IIF):
wherein R1 and q ar
Figure imgf000119_0002
[0277] In some embodiments, the compound of Formula (II) is a compound of the Formula (IIG):
Figure imgf000119_0003
wherein R1 and q are as defined herein.
[0278] In some embodiments, R1 is, independently, alkyl or halo. In some embodiments, R1 is, independently, C1-6 alkyl, C1-6 haloalkyl, or halo. In some embodiments, R1 is, independently, C1-6 haloalkyl or halo. In some embodiments, R1 is, independently, halo. In some embodiments, R1 is, independently, fluoro, chloro, bromo, iodo, or m, p, or q is 0 or 1. In some embodiments, n, m, p, or q is 0.
[0279] In some embodiments, R1 is, independently, alkyl, alkoxy, heteroalkyl, halo, haloalkyl, or haloalkoxy. In some embodiments, R1 is, independently, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, C1-6 haloalkoxy, or halo. In some embodiments, R1 is, independently, C1-6 alkyl or C1-6 alkoxy. In some embodiments, R1 is, independently, alkoxy. In some embodiments, R1 is, independently, methoxy, ethoxy, propoxy. In some embodiments, n, m, p, or q is 0, 1 or 2. In some embodiments, n, m, p, or q is 0 or 1. In some embodiments, n, m, p, or q is 0.In some embodiments, the compound of Formula (II) is a compound of Formula (IIA):
Figure imgf000120_0001
wherein:
R1 is, independently at each occurrence, halo or trifluoromethyl; and n is 0, 1, or 2.
[0280] In some embodiments, the compound of Formula (II) is a compound of Formula (IIB):
Figure imgf000120_0002
wherein:
R1 is, independently at each occurrence, halo or trifluoromethyl; and q is 0, 1, or 2.
[0281] In some embodiments, the compound of Formula (II) is:
,
Figure imgf000121_0001
from
,
,
.
[0
Figure imgf000122_0001
.
[0284] In certa
Figure imgf000122_0002
the payload portion of the conjugates described herein and are released, e.g., by enzyme proteolysis, following internalization of the conjugate into a cell. The methods provided herein include methods of treating a proliferative disease, e.g., cancer, comprising administering to a patient a therapeutically effective amount of a conjugate, e.g., antibody-drug conjugate that releases a patient.
[0285] In some embodiments, these compounds represent the metabolic product of the conjugates described herein, e.g., enzyme proteolysis product.
[0286] In some embodiments, A is a divalent radical of benzene, of pyridine, of naphthalene, or of quinolone, which are optionally substituted.
[0287] In some embodiments, A is arylene.
[0288] In some embodiments, A is:
,
wherein:
Figure imgf000123_0001
R1 is, independently at each occurrence, alkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl, halo, haloalkoxy, heteroaryl, heterocycloalkyl, hydroxyl, cyano, nitro, , , , or azido,
Figure imgf000123_0004
Figure imgf000123_0003
n is an integer from 0 to 4;
m is and integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5.
[0289] In some embodiments, the compound of Formula (II) is a compound of the Formula (IIA):
Figure imgf000123_0002
wherein R1 is, independently at each occurrence, methoxy, halo or trifluoromethyl; and n is 0, 1, or 2. Formula (IIB):
Figure imgf000124_0001
wherein R1 is, independently at each occurrence, methoxy, halo or trifluoromethyl; and q is 0, 1, or 2. [0291] In some embodiments, the compound of Formula (II) is a compound of the Formula (IIB2):
Figure imgf000124_0002
wherein R1 is, independently at each occurrence, methoxy, halo or trifluoromethyl; and q is 0, 1, or 2. [0292] In some embodiments, the compound of Formula (II) is a compound of the Formula (IIB3):
Figure imgf000125_0001
[0293] wherein R1 is, independently at each occurrence, methoxy, halo or trifluoromethyl; and q is 0, 1, or 2.In some embodiments, R1 is, independently, alkyl or halo. In some embodiments, R1 is, independently, C1-6 alkyl, C1-6 haloalkyl, or halo. In some embodiments, R1 is, independently, C1-6 haloalkyl or halo. In some embodiments, R1 is, independently, halo. In some embodiments, R1 is, independently, fluoro, chloro, bromo, iodo, or trifluoromethyl. In some embodiments, n, m, p, or q is 0, 1 or 2. In some embodiments, n, m, p, or q is 0 or 1. In some embodiments, n, m, p, or q is 0.
[0294] In some embodiments, R1 is, independently, alkyl, alkoxy, heteroalkyl, halo, haloalkyl, or haloalkoxy. In some embodiments, R1 is, independently, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, C1-6 haloalkoxy, or halo. In some embodiments, R1 is, independently, C1-6 alkyl or C1-6 alkoxy. In some embodiments, R1 is, independently, alkoxy. In some embodiments, R1 is, independently, methoxy, ethoxy, propoxy. In some embodiments, n, m, p, or q is 0, 1 or 2. In some embodiments, n, m, p, or q is 0 or 1. In some embodiments, n, m, p, or q is 0.
[0295] In some embodiments, the compound of Formula (II) is:
,
Figure imgf000125_0002
,
Figure imgf000126_0001
Formua IIH:
Figure imgf000127_0001
wherein R1 and n are as defined herein.
[0297] In some embodiments, the compound of Formula (II) is a compound selected from CH 3 CH3 CH
H OH O H OH OCH 3 3
O N O N
,
,
Figure imgf000127_0002
,
Figure imgf000128_0001
[0299] Compounds of Formula I can be synthesized by coupling compounds of Formula P1 with a binding agent, e.g., antibody under standard conjugation conditions (see, e.g, Doronina et al., Nature Biotechnology 2003, 21, 7, 778, which is incorporated herein by reference). When the binding agent is an antibody, the antibody can be coupled to a compound of Formula P1 via one or more cysteine or lysine residues of the antibody.
Compounds of Formula P1 can be coupled to cysteine residues, for example, by subjecting the antibody to a reducing agent, e.g., dithiotheritol, to cleave the disulfide bonds of the antibody, purifying the reduced antibody, e.g., by gel filtration, and subsequently reacting the antibody with a compound of formula P1 containing a reactive moiety, e.g., a maleimido group. Suitable solvents include, but are not limited to water, DMA, DMF, and DMSO. Compounds of formula P1 containing a reactive moiety, e.g., activated ester or acid halide group, can be coupled to lysine residues. Suitable solvents include, but are not limited to protein techniques, including, for example, size exclusion chromatography, dialysis, and ultrafiltration/diafiltration.
Figure imgf000129_0001
wherein RL is a reactive linker, A is arylene or heteroarylene, L is a linker, and BA is a binding agent.
[0300] In some embodiments, the compound of formula P1 includes A, wherein A is:
Figure imgf000129_0002
Figure imgf000130_0001
[0303] The reactive linker is a moiety comprising a portion in its structure that is capable of reacting with the binding agent (e.g., reacting with an antibody at its cysteine or lysine residues) to form the compound of Formula I. Following conjugation to the binding agent, the reactive linker becomes the linker (L) moiety of the compound of Formula I. Illustrative reactive linkers include, but are not limited to, those that comprise haloacetyl, isothiocyanate, or maleimide portions that are capable of reacting with the binding agent. Reactive portions also include moieties having the following structure:
Figure imgf000130_0002
[0304] In some embodiments, the reactive linker is: wherein:
SPR is a reactive spacer;
AA1 is an amino acid; and
AA2 is an amino acid.
[0305] The reactive spacer is a moiety that contains the above-described reactive linker portion that is capable of reacting with the binding agent and connects this portion to AA1. Suitable spacers include, but are not limited to, those comprising alkylene or polyethylene glycol connecting the AA1 to the portion capable of reacting with binding agent (e.g., haloacetyl, isothiocyanate, or maleimide).
[0306] In some embodiments, the reactive spacer comprises a non-cleavable moiety selected from
Figure imgf000131_0001
examples, n is 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the reactive spacer is
In some embodiments, the reactive spacer is
.
Figure imgf000131_0002
mbodiments, the reactive spacer is:
, wherein b is an integer
Figure imgf000131_0003
[0309] In some em o ments, t e react ve spacer s:
[0310] In .
Figure imgf000131_0004
[0311] In some embodiments, the spacer or
Figure imgf000131_0005
, wherein g is an integer from 1 to 24.
Figure imgf000131_0006
e embodiments, the reactive spacer is: wherein b is an
Figure imgf000132_0001
[0313] In some embodiments, the reactive spacer is:
. [0314]
Figure imgf000132_0002
, , aline, lysine- phenylalanine, phenylalanine-lysine, valine-asparagine, asparagine-valine, threonine- asparagine, asparagine-threonine, serine-asparagine, asparagine-serine, phenylalanine- asparagine, asparagine-phenylalanine, leucine-asparagine, asparagine-leucine, isoleucine- asparagine, asparagine-isoleucine, glycine-asparagine, asparagine-glycine, glutamic acid- asparagine, asparagine-glutamic acid, citrulline-asparagine, asparagine-citrulline, alanine- asparagine, or asparagine-alanine.
[0315] In some embodiments, AA1-AA2 is: valine-citrulline or citrulline-valine. In some embodiments, AA1-AA2 is: valine-citrulline.
[0316] In some embodiments, the reactive linker is:
wherein:
Figure imgf000132_0003
SPR is a reactive spacer;
RAA1 is an amino acid side chain; and
RAA2 is an amino acid side chain.
[0317] In some embodiments, the reactive linker is:
Figure imgf000132_0004
SP is a reactive spacer.
[0318] In some embodiments, the reactive linker is:
2
wherein b is an integer
Figure imgf000133_0001
[0319] In some embodiments, the reactive linker is:
wherein b is an integer
Figure imgf000133_0002
[0320] In some embodiments, the reactive linker is:
Figure imgf000133_0003
wherein b is an integer from 2 to 8.
[0321] In some embodiments, the reactive linker is:
wherein b is an integ
Figure imgf000133_0004
[0322] In some embodiments, the reactive linker is: wherein b is an inte
Figure imgf000134_0001
, , d RM is alkyl. [0323] In some embodiments, the reactive linker is:
O NH2
wherein b is an integ
Figure imgf000134_0002
[0324] In some embodiments, the reactive linker is:
2
wherein b is an integ
Figure imgf000134_0003
[0325] In some embodiments, the reactive linker is:
wherein b is an inte d RM is alkyl.
Figure imgf000134_0004
[0326] In some embodiments, the reactive linker is: . wherein b is an integ
Figure imgf000135_0001
[0327] In some embodiments, the reactive linker is:
wherein g is an int
Figure imgf000135_0002
. [0328] In some embodiments, the reactive linker is:
Figure imgf000135_0003
.
[0329] In some embodiments, the reactive linker is:
.
Figure imgf000135_0004
[0330] In some embodiments, the reactive linker is: [0331] In
Figure imgf000136_0005
some embodiments, the reactive linker is:
.
[0332] In
Figure imgf000136_0001
.
[0333] In
Figure imgf000136_0002
.
[0334] In
Figure imgf000136_0003
.
[0335] In
Figure imgf000136_0004
.
[0336] In
Figure imgf000137_0001
.
Figure imgf000137_0002
[0337] In some embodiments, the reactive linker is:
.
[0338] In
Figure imgf000137_0003
O NH2
.
[0339]
Figure imgf000137_0004
.
Figure imgf000137_0005
[0340] In some embodiments, the compound of Formula P1 is a compound of Formula P1A:
Figure imgf000138_0001
wherein:
A is:
,
Figure imgf000138_0002
R is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heterocycloalkyl, hydroxyl, cyano, nitr , or azido, wherein RA is alkyl
Figure imgf000138_0003
n is an integer rom 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5;
SPR is a reactive spacer;
AA1 is an amino acid; and
AA2 is an amino acid. [0341] In some embodiments, the compound of Formula P1A is a compound which includes A wherein A is:
Figure imgf000138_0004
methylsulfonyl, N-methylformamide, hydroxyl, or morpholinyl.
[0342] In some embodiments, the compound of Formula P1A is a compound which includes A wherein A is:
,
wherein n is 0 or 1; and R1 is C1-6 alko
Figure imgf000139_0001
1-6 haloalkyl.
[0343] In some embodiments, the compound of Formula P1A is a compound which includes A wherein A is:
,
wherein q is an integer from 0 to 5; and
Figure imgf000139_0002
alkoxy, halo, or C1-6 haloalkyl.
[0344] In some embodiments, the compound of Formula P1A is a compound which includes A wherein A is:
,
wherein q is an integer from 0 to 5; and
Figure imgf000139_0003
alkoxy, halo, or C1-6 haloalkyl.
[0345] In some embodiments, the compound of Formula P1A is a compound which includes A wherein A is:
,
wherein n is 0 or 1; and R1 is alkoxy, ha
Figure imgf000139_0004
alkyl. In some examples, R1 is methylsulfonyl, N-methylformamide, hydroxyl, or morpholinyl.
[0346] In some embodiments, the compound of Formula P1 is a compound of Formula P1B:
Figure imgf000140_0001
wherein
A is:
,
Figure imgf000140_0002
R s a y, a eny, a yny, a oxy, ary, alkaryl, aralkyl, halo, haloalkoxy, haloalkyl, heteroaryl, heterocycloalkyl, hydroxyl, cyano nitro or azido wherein
Figure imgf000140_0004
n is an integer from 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5; and
SPR is a reactive spacer.
[0347] In some embodiments, the compound of Formula P1 is a compound of Formula P1C:
Figure imgf000140_0003
wherein: AA1 is an amino acid;
AA2 is an amino acid;
R1 is, independently at each occurrence, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl, and
n is 0, 1, or 2.
[0348] In some embodiments, the compound of Formula P1 is a compound of Formula P1D:
Figure imgf000141_0001
wherein:
SPR is a reactive spacer;
AA1 is an amino acid;
AA2 is an amino acid;
R1 is, independently at each occurrence, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl, and
n is 0, 1, or 2. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2.
[0349] In some embodiments, the compound of Formula P1 is a compound of Formula P1D, wherein R1 is alkoxy, halo, or haloalkyl. In some embodiments, R1 is C1-6 alkoxy, halo, or C1-6 haloalkyl. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2.
[0350] In some embodiments, the compound of Formula P1 is a compound of Formula P1D, wherein R1 is C1-6 alkoxy, halo, or C1-6 haloalkyl; and SPR-AA1-AA2 is , wherein b is an integer from 2 to 8 and is a bodiments, n is 0. In some embodiments, n is 1. In
Figure imgf000142_0001
some embodiments, n is 2. In some embodiments b is 2. In some embodiments b is 3. In some embodiments b is 4. In some embodiments b is 5. In some embodiments b is 6. In some embodiments b is 7. In some embodiments b is 8.
[0351] In some embodiments, the compound of Formula P1 is a compound of Formula P1E:
Figure imgf000142_0002
wherein:
SPR is a reactive spacer;
RAA1 is an amino acid side chain;
RAA2 is an amino acid side chain;
R1 is, independently at each occurrence, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl, and
n is 0, 1, or 2.
[0352] In some embodiments, the compound of Formula P1 is a compound of Formula P1F:
Figure imgf000143_0001
wherein:
SPR is a reactive spacer;
R1 is, independently at each occurrence, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl, and
n is 0, 1, or 2.
[0353] In some embodiments, the compound of Formula P1 is a compound of Formula P1G:
H H 3 CH
OH OC 3
O N 3
Figure imgf000143_0002
wherein:
SPR is a reactive spacer; and
R1 is a hydrogen atom, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl.
[0354] In some embodiments, the compound of Formula P1 is a compound of Formula P1H:
Figure imgf000144_0001
wherein:
R1 is hydrogen atom, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl; and
b is an integer from 2 to 8.
[0355] In some embodiments, the compound of Formula P1 is a compound of Formula P1I:
H CH 3 CH
OH O 3 O N 3
wher
Figure imgf000144_0002
R1 is a hydrogen atom, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl; and
b is an integer from 2 to 8.
[0356] In some embodiments, the compound of Formula P1 is a compound of Formula P1J:
Figure imgf000145_0001
wherein:
R1 is a hydrogen atom, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl; and
b is an integer from 2 to 8.
[0357] In some embodiments, the compound of Formula P1 is a compound of Formula P1K:
Figure imgf000145_0002
weren s a yrogen atom, a y, a oxy, ao, aoa oxy, aoa y, or tr uoromethyl.
[0358] In some embodiments, the compound of Formula P1 is a compound of Formula P1L:
Figure imgf000145_0003
[0359] In some embodiments, the compound of Formula P1 is a compound of Formula P1M:
Figure imgf000146_0001
wherein R is a hydrogen atom, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl.
[0360] In some embodiments, the compound of Formula P1 is a compound of Formula P1N:
Figure imgf000146_0002
wherein R is a hydrogen atom, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl, and b is an integer from 2 to 8.
[0361] In some embodiments, the compound of Formula P1 is a compound of Formula P1O:
Figure imgf000146_0003
wherein R1 is a hydrogen atom, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl.
[0362] In some embodiments, the compound of Formula P1 is a compound of Formula P1P:
Figure imgf000147_0001
weren R s a yrogen atom, a y, a oxy, ao, aoa oxy, aoa y, or tr uoromethyl, and b is an integer from 2 to 8.
[0363] In some embodiments, the compound of Formula P1 is a compound of Formula P1Q:
Figure imgf000147_0002
wherein R is a hydrogen atom, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl, and b is an integer from 2 to 8.
[0364] In some embodiments, the compound of Formula P1 is a compound of Formula P1R:
Figure imgf000148_0001
[0365] In some embodiments, the compound of Formula P1 is a compound of Formula P1S:
Figure imgf000148_0002
wherein R is a hydrogen atom, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl.
[0366] In some embodiments, the compound of Formula P1 is a compound of Formula P1T:
Figure imgf000148_0003
wherein RN is a hydrogen atom or alkyl, RM is alkyl, R1 is a hydrogen atom, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl, and b is an integer from 2 to 8. Formula P1U:
Figure imgf000149_0001
wherein RN is a hydrogen atom or alkyl, RM is alkyl, R1 is a hydrogen atom, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl, and b is an integer from 2 to 8.
[0368] In some embodiments, the compound of Formula P1 is a compound of Formula P1V:
Figure imgf000149_0002
wherein R1 is a hydrogen atom, alkyl, alkoxy, halo, haloalkoxy, haloalkyl, or trifluoromethyl, and b is an integer from 2 to 8.
[0369] In some embodiments, the compound of Formula P1 is a compound of Formula P1W:
Figure imgf000149_0003
g is an integer from 2 to 20; and b is an integer from 2 to 8.
[0370] In some embodiments, the compound of Formula P1 is a compound of Formula P1X:
Figure imgf000150_0001
wherein:
R1 is alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, halo, haloalkyl, or haloalkoxy; and b is an integer from 2 to 8. In some embodiments, R1 is methyl, ethyl, methoxy, or ethoxy. In some of these embodiments, R1 is methoxy. In some embodiments, R1 is 1-methylethyl-thiol, phenyl, 2-fluorophenyl, pyridinyl, 4-pyridinyl, pyrrolidinyl, or 1-pyrrolidinyl. In some embodiments, R1 is trifluoromethyl. In some embodiments, R1 is fluoro. In some embodiments, R1 is hydrogen.
[0371] In some embodiments, the compound of Formula P1 is a compound of Formula P1Y:
C H OCH 3 H
H 3
3
Figure imgf000150_0002
weren R s a y, a eny, a yny, a oxy, eteroa y, ao, aoa y, aoa oxy. In some embodiments, R1 is methyl, ethyl, methoxy, or ethoxy. In some of these embodiments, R1 is methoxy. In some embodiments, R1 is 1-methylethyl-thiol, phenyl, 2-fluorophenyl, pyridinyl, 4-pyridinyl, pyrrolidinyl, or 1-pyrrolidinyl. In some embodiments, R1 is trifluoromethyl. In some embodiments, R1 is fluoro. In some embodiments, R1 is hydrogen. Formula P1Z:
Figure imgf000151_0001
wherein Rc is selected from alkyl or haloalkyl and wherein the alkyl or haloalkyl is linear, branched, or cyclic.
[0373] In some embodiments, the compound of Formula P1 is a compound having one of the following structures:
,
,
Figure imgf000151_0002
,
, , ,
Figure imgf000152_0001
,
Figure imgf000153_0001
,
,
Figure imgf000154_0001
,
,
Figure imgf000155_0001
,
,
Figure imgf000156_0001
,
, , ,
Figure imgf000157_0001
,
,
Figure imgf000158_0001
,
Figure imgf000159_0001
O
3 ,
Figure imgf000160_0001
,
,
Figure imgf000161_0001
Figure imgf000162_0001
,
,
Figure imgf000163_0001
,
Figure imgf000164_0001
Figure imgf000165_0001
Figure imgf000166_0001
Figure imgf000167_0001
Figure imgf000168_0001
Figure imgf000169_0001
,
,
Figure imgf000170_0001
Figure imgf000171_0001
Figure imgf000172_0001
, , , ,
Figure imgf000173_0001
,
Figure imgf000174_0001
,
,
Figure imgf000175_0001
,
, , ,
Figure imgf000176_0001
,
Figure imgf000177_0001
,
Figure imgf000178_0001
,
Figure imgf000179_0001
,
,
Figure imgf000180_0001
,
, , ,
Figure imgf000181_0001
,
,
Figure imgf000182_0001
, , , ,
Figure imgf000183_0001
3
,
Figure imgf000184_0001
Figure imgf000185_0001
, ,
Figure imgf000185_0002
,
, , ,
Figure imgf000186_0001
Figure imgf000187_0001
, , , ,
Figure imgf000188_0001
,
3 ,
Figure imgf000189_0001
,
,
Figure imgf000190_0001
,
, , ,
Figure imgf000191_0001
,
,
Figure imgf000192_0001
, , , ,
Figure imgf000193_0001
,
Figure imgf000194_0001
,
,
Figure imgf000195_0001
Figure imgf000196_0001
,
Figure imgf000197_0001
,
Figure imgf000198_0001
,
Figure imgf000199_0001
,
,
Figure imgf000200_0001
,
, , ,
Figure imgf000201_0001
,
, ,
Figure imgf000202_0001
, , , ,
Figure imgf000203_0001
,
,
Figure imgf000204_0001
,
, , ,
Figure imgf000205_0001
,
, ,
Figure imgf000206_0001
,
, , ,
Figure imgf000207_0001
Figure imgf000208_0001
,
Figure imgf000209_0001
.
Figure imgf000210_0001
d of Formula P1 is a compound having one of the following structures:
,
,
Figure imgf000210_0002
,
, or
.
Figure imgf000211_0001
g compounds of Formula P2 with the compound of Formula P3 under amide synthesis conditions. Suitable amide synthesis conditions include, but are not limited to, contacting the compound of Formula P2 in the presence of a carboxylic acid activating agent and base. Suitable activating agents include, but are not limited to EDC, HATU, HBTU, DCC, BOP, and EEDQ. Suitable bases include, but are not limited to DIEA, DBU, Tributylamine, and 2,6- Lutidine. and alanine using known techniques (see, e.g., U.S. Patent No.4,308,269, which is incorporated herein by reference).
Figure imgf000212_0001
ompouns o ormua can e syn esze y coup ng compouns o Formula PP3:
Figure imgf000212_0002
with compounds of Formula PP4 under amide synthesis conditions:
wherein:
Figure imgf000212_0003
BA is a binding agent;
SP is a spacer;
RAA1 is an amino acid side chain;
RAA2 is an amino acid side chain; k is an integer from 1 to 10.
[0378] Compounds of Formula PP3 can be synthesized by contacting compounds of Formula PP5 with a suitable reducing agent:
Figure imgf000213_0001
wherein A is arylene or heteroarylene.
[0379] In some embodiments, the suitable reducing agent includes a metal, a metal foil, a metal powder, a metal amalgam, or metal filings. In certain embodiments, the metal is selected from zinc , iron, aluminum, palladium, or Raney nickel.
[0380] For example, in some embodiments, the following reducing agent conditions are employed. With respect to the amount of compound PP5, for example, in some of the methods herein about twenty (20) equivalents of zinc dust and forty (40) equivalents of acetic acid were combined. In some examples, the reducing reaction was conducted at room temperature for about from 1 to 20 hours. In some of these examples, the aforementioned acetic acid is substituted with another suitable mild acid or proton donor. Examples of suitable mild acids or proton donors include, but are not limited to formic acid, pTsOH, and NH4Cl. In some of these examples, the reducing metal is substituted with a suitable reducing agent selected from iron, aluminum, palladium, or Raney nickel. In some of these examples, suitable solvents includes those solvents having 10-50 % water (by volume) in a miscible organic solvent. Example miscible organic solvents include, but are not limited to THF, Dioxane, and diethyl ether. In some examples, the reducing reactions set forth herein are conducted at reaction temperatures which range from 0 to 50°C. In some examples, the reducing reactions set forth herein are conducted at reaction times which range from 1 to 40 hours.
[0381] Suitable acids include, but are not limited to, acetic acid.
[0382] In some embodiments, A is: ,
wherein:
Figure imgf000214_0001
R1 is, independently at each occurrence, alkyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl, halo, heteroaryl, heterocycloalkyl, hydroxyl, cyano, nitro, , ,
wherein R is alkyl or heteroalkyl;
Figure imgf000214_0003
n is an integer from 0 to 4;
m is and integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5.
[0383] In some embodiments, the compound of Formula PP5 is a compound of the Formula PP5A:
Figure imgf000214_0002
wherein R1 and n are as defined herein.
[0384] In some embodiments, R1 is, independently, alkyl, alkoxy, heteroalkyl, halo, haloalkyl, or haloalkoxy. In some embodiments, R1 is, independently, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, C1-6 haloalkoxy, or halo. In some embodiments, R1 is, independently, C1-6 alkyl or C1-6 alkoxy. In some embodiments, R1 is, independently, alkoxy. In some embodiments, R1 is, independently, methoxy, ethoxy, propoxy. In some embodiments, n, m, p, or q is 0, 1 or 2. In some embodiments, n, m, p, or q is 0 or 1. In some embodiments, n, m, p, or q is 0.
[0385] In some embodiments, the compound of Formula PP5 is a compound of the Formula PP5A:
Figure imgf000215_0001
wherein:
R1 is, independently at each occurrence, halo or trifluoromethyl; and n is 0, 1, or 2.
[0386] In some embodiments, the compound of Formula PP5 is a compound of the Formula PP5A2:
Figure imgf000215_0002
wherein:
R1 is, independently at each occurrence, halo or trifluoromethyl; and q is an integer from 0 to 5
[0387] In some embodiments, the compound of Formula PP5 is a compound of the Formula PP5A3:
Figure imgf000215_0003
R1 is, independently at each occurrence, halo or trifluoromethyl; and q is an integer from 0 to 5.In some embodiments, R1 is 1-methylethyl-thiol, phenyl, 2- fluorophenyl, pyridinyl, 4-pyridinyl, pyrrolidinyl, or 1-pyrrolidinyl. In some embodiments, R1 is trifluoromethyl. In some embodiments, R1 is methoxy. In some embodiments, R1 is fluoro. In some embodiments, R1 is hydrogen.
[0388] In some embodiments, the compound of Formula PP5 is a compound of the Formula PP5A4:
Figure imgf000216_0001
wherein R1 and n are as defined herein.
[0389] Compounds of Formula PP5 can be synthesized by contacting compounds of Formula P2 with compounds of Formula PP6 under amide synthesis conditions:
Figure imgf000216_0002
Figure imgf000216_0003
[0390] Suitable compounds of Formula PP6 include, but are not limited to, 3-nitro- benzoic acid, 3-chloro-5-nitro-benzoic acid, 3-fluoro-5-nitro-benzoic acid, 3-nitro-1- naphthalenecarboxylic acid, 2-fluoro-5-nitro-benzoic acid, 3-(dimethylamino)-5-nitro- benzoic acid, 3-ethoxy-5-nitro-benzoic acid, 2-methoxy-5-nitro-benzoic acid, 4-methoxy-3- 6-chloro-2-fluoro-3-nitro-benzoic acid, 2-chloro-4-fluoro-5-nitro-benzoic acid, 4-chloro-2- fluoro-5-nitro-benzoic acid, 2-ethoxy-5-nitro-benzoic acid, 2-(methylamino)-3-nitro-benzoic acid, 6-nitro-8-quinolinecarboxlic acid, 4-(dimethylamino)-3-nitro-benzoic acid
hydrochloride (1:1), 2-methyl-nitro-benzoic acid, 3-methyl-4-nitro-benzoic acid, 4-nitro-1- naphthalenecarboxylic acid, 4-nitro-1-naphthalenecarboxylic acid, 2,6-dimethyl-4-nitro- benzoic acid, 3-fluoro-4-nitro-benzoic acid, 3-chloro-4-nitro-benzoic acid, 3-bromo-4-nitro- benzoic acid, 3-cyano-4-nitro-benzoic acid, 3-cyclopropyl-4-nitro-benzoic acid, 3-methoxy- 4-nitro-benzoic acid, 2-methoxy-4-nitro-benzoic acid, 5-chloro-2-methyl-4-nitro-benzoic acid, 8-nitro-5-isoquinolinecarboxylic acid, 5-nitro-8-quinolinecarboxylic acid, 8-nitro-5- quinolinecarboxylic acid, 2,5-difluoro-4-nitro-benzoic acid, 2-(dimethylamino)-4-nitro- benzoic acid,, 2-chloro-5-fluoro-4-nitro-benzoic acid, 3-(dimethylamino)-4-nitro-benzoic acid, 2-[(1-methylethyl)thio]-4-nitro-benzoic acid, 4-nitro-3-(trifluoromethyl)-benzoic acid, 4-nitro-2-(trifluoromethyl)-benzoic acid, 3,5-dimethoxy-4-nitro-benzoic acid, 4-nitro-2- (propylamino)-benzoic acid, 3-(difluoromethoxy)-4-nitro-benzoic acid, 2-(2-fluoro-phenyl)- 4-nitro-benzoic acid, 4-nitro-2-(4-pyridinyl)-benzoic acid, 4-nitro-3-(4-pyridinyl)-benzoic acid, or 4-nitro-2-(1-pyrrolidinyl)-benzoic acid.
[0391] Suitable compounds of Formula PP6 include compounds having any one of the following formula:
r
w lkyl,
Figure imgf000217_0001
or C1-6 haloalkoxy, wherein n is 0, 1, 2, 3, or 4. In certain of these embodiments, R is methoxy or methyl. In some specific embodiments, R1 is methoxy, fluoro, or trifluoromethyl. In certain embodiments, n is 1 or 2. In some of these embodiments, n is 1. In some embodiments, R1 is fluoro, chloro, bromo, or iodo. pyridinyl, 4-pyridinyl, pyrrolidinyl, or 1-pyrrolidinyl. In some embodiments, R1 is trifluoromethyl. In some embodiments, R1 is methoxy. In some embodiments, R1 is fluoro. In some embodiments, R1 is hydrogen.
[0393] In some embodiments, provided herein are compounds of Formula PP5:
Figure imgf000218_0001
wherein A is arylene or heteroarylene.
[0394] In some embodiments, the compound of Formula PP5 is a compound selected from
,
,
.
Figure imgf000219_0001
.
[0396] Compo
Figure imgf000219_0002
Figure imgf000220_0001
can be synthesized by contacting compounds of Formula PP1:
Figure imgf000220_0002
with a binding agent un er con ugat on con t ons, wherein:
BA is a binding agent;
SP is a spacer;
SPR is a reactive spacer;
RAA1 is an amino acid side chain; RAA2 is an amino acid side chain; A is arylene or heteroarylene; and k is an integer from 1 to 30.
Formula PP2 with the compound of Formula P2:
Figure imgf000221_0001
wherein:
SPR is a reactive linker;
RAA1 is an amino acid side chain;
RAA1 is an amino acid side chain; and
A is arylene or heteroarylene.
[0398] Compounds of Formula PP1 can be prepared by contacting a compound of Formula PP3 with the compound of Formula PP7:
Figure imgf000221_0002
SP is a reactive linker;
RAA1 is an amino acid side chain;
RAA1 is an amino acid side chain; and
A is arylene or heteroarylene.
[0399] Compounds of Formula PP2 can be prepared by contacting a compound of Formula PP8 with a bifunctional spacer: .
Figure imgf000222_0001
[0400] Compounds of Formula PP7 can be prepared by contacting a compound of Formula PP9 with a bifunctional spacer:
.
Figure imgf000222_0002
[0401] Bifunctional spacers are compounds that react with the compound of Formula PP3 to append the SPR moiety present in the compounds of Formula PP2. Illustrative bifunctional spacers include, but are not limited to:
.
Figure imgf000222_0003
[0402] Compounds of Formula PP8 can be prepared by contacting a compound of Formula PP10 with a compound of Formula PP11, following by removal of the protecting group:
O
1 H2N H
wherei
Figure imgf000222_0004
l to which it is attached electrophilic. Compound of Formula PP10 can be prepared by coupling its corresponding amino acids using standard amino acid coupling techniques, including, for example, active ester formation using HATU, BOP/HOBt, or EDC/N-hydroxysuccinamide in the presence of DIEA, DBU, or tributylamine. PP9 to append the SPR moiety present in the compounds of Formula PP7. Illustrative bifunctional spacers include, but are not limited to:
,
,
.
Figure imgf000223_0001
[0404] Antibody-drug-conjugate compounds of Formula (I) can also be prepared by reacting a suitable antibody, e.g., deglycosylated antibody or aglycosylated antibody with a compound of Formula (PT1) in the presence of transglutaminase:
Figure imgf000223_0002
A is arylene or heteroarylene; and
L is a linker.
[0405] In some embodiments, A is:
,
wherein:
Figure imgf000224_0001
R1 is, independently at each occurrence, halo, haloalkyl, haloalkoxy, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, aryl, alkaryl, aralkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano, nitr , or azido,
wherein RA is alkyl o
Figure imgf000224_0002
n is an integer from 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5.
[0406] In some embodiments, A is:
wherein:
Figure imgf000224_0003
R1 is, independently at each occurrence, halo, haloalkyl, hydroxyl, alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, heteroaryl, heteroalkyl, heterocycloalkyl, cyano nitro or azido
Figure imgf000224_0004
n is an integer from 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5.
[0407] In some embodiments, R1 is, independently, alkyl or halo. In some embodiments, R1 is, independently, C1-6 alkyl, C1-6 haloalkyl, or halo. In some embodiments,
Figure imgf000225_0002
methylformamide. In some embodiments, R1 is morpholinyl.
[0409] In some embodiments, the linker comprises one or more amino acids. Suitable amino acids include natural, non-natural, standard, non-standard, proteinogenic, non- proteinogenic, and L-, or D- ^ ^ amino acids. In some embodiments, the linker comprises alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, or derivative thereof.
[0410] In some embodiments, the linker comprises valine and citrulline.
[0411] In some embodiments, the linker is:
Figure imgf000225_0001
AA2 is an amino acid.
[0412] The linker may further comprise a divalent moiety that connects the AA1-AA2 moiety to the–NH2 of PT1. Suitable divalent moieties include, but are not limited to, those comprising alkylene or polyethylene glycol. The divalent moitety may comprise one or more reactive groups to facilitate bonding to the rest of the compound, or one or more residues of such reactive groups.
[0413] PT1 includes a primary amine-terminated alkylene or a primary amine- terminated polyethylene glycol. The primary amine-terminating moiety can be directly bonded to a deglycosylated antibody or aglycosylated antibody in the presence of transglutaminase.
[0414] In some embodiments, the compound comprises a primary amine-terminated alkylene. In some embodiments, the compound comprises a NH2-C5-7 alkylene. In some embodiments, the compound comprises:
Figure imgf000226_0003
wherein:
is a bond to the payload; and
Figure imgf000226_0004
is an integer from 2 to 8.
[0415] In some embodiments, the compound comprises: H2N
.
wherein:
Figure imgf000226_0001
is a bond to the payload.
Figure imgf000226_0005
[0416] In some embodiments, the compound of PT1 is
.
[0417]
Figure imgf000226_0002
d by contacting a binding agent with PT1 in the presence of transglutaminase under conditions suitable for a transglutamination reaction. In some embodiments, the transglutaminase reaction is at a pH between about 7 and about 8 for at least 4 hr. In some examples, the pH is 7.2, 7.3, 7.4, 7.5, 7.6, 7.8, or 8.
[0418] In some embodiments, the compound of Formula (I) is prepared by a transglutaminase reaction wherein the concentration of the compound of Formula (PT1) is at a concentration of at least 30 molar equivalents compared to the deglycosylated antibody or aglycosylated antibody. In some embodiments, the compound of Formula (I) is prepared by a transglutaminase reaction wherein the concentration of the compound of Formula (PT1) is at a concentration of 30 to 150 molar equivalents compared to the deglycosylated antibody or aglycosylated antibody. transglutaminase reaction wherein the concentration of the compound of Formula (PT1) is 1 to 30 U per milligram of deglycosylated antibody or aglycosylated antibody.
[0420] In some embodiments, the antibody is deglycosylated with peptide N- glycosidase F (PNGaseF) prior to the transglutaminase reaction.
[0421] In some embodiments, the antibody is aglycosylated. An aglycosylated antibody can be prepared by mutagenesis techniques to remove one or more amino acid sequences that are necessary for glycosylation of the antibody. In certain embodiments the antibody comprises a heavy chain with a mutation that substitutes another amino acid for N180. In certain embodiments, the aglycosylated antibody comprises one or more N180Q heavy chain polypeptides.
[0422] In some embodiments, the compound of Formula (I) is prepared by a transglutaminase reaction which is conducted in one or more solvent(s) selected from the group consisting of water, buffered water, saline water, buffered saline water, and an organic.
[0423] In some embodiments, the compound of Formula (I) is prepared by a transglutaminase reaction which is conducted in water buffered with phosphate, HEPES, or MOPS.
[0424] In some embodiments, the compound of Formula (I) is prepared by a transglutaminase reaction which includes reacting the glutaminyl-modified antibody with a reactive spacer compound to form an antibody-spacer conjugate; and then reacting the antibody-spacer conjugate with a reactive payload compound to form an antibody-spacer- payload conjugate.
[0425] In some embodiments, provided herein is a glutaminyl-modified antibody produced by a method set forth herein.
[0426] In some embodiments, provided herein is a pharmaceutical composition comprising a glutaminyl-modified antibody produced by a method set forth herein.
[0427] In some embodiments, provided herein is a method of treating a condition in a subject in need thereof comprising administering to the subject a pharmaceutically acceptable amount of the antibody or antibody-drug-conjugate provided herein.
[0428] In some embodiments, provided herein is an antibody or antibody-drug- conjugate described herein for therapy.
[0429] In some embodiments, provided herein is an antibody or antibody-drug- conjugate described herein for the treatment of cancer. [0430] The present disclosure includes methods of treating or preventing diseases, conditions, or disorders e.g., proliferative diseases such as cancer, comprising administering a therapeutically effective amount or one or more of the compounds disclosed herein, e.g., one or more of the compounds of Formula (I) or (II). Diseases, disorders, and/or conditions include, but are not limited to, those associated with the antigens listed herein. In some embodiments, the antigen is PSMA, MUC16, or EGFRvIII.
[0431] The compounds disclosed herein can be used for treating primary and/or metastatic tumors arising in the brain and meninges, oropharynx, lung and bronchial tree, gastrointestinal tract, male and female reproductive tract, muscle, bone, skin and appendages, connective tissue, spleen, immune system, blood forming cells and bone marrow, liver and urinary tract, and special sensory organs such as the eye. In certain embodiments, the compounds provided herein are used to treat one or more of the following cancers: renal cell carcinoma, pancreatic carcinoma, head and neck cancer, prostate cancer, malignant gliomas, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non- small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer, or melanoma. In some embodiments, the cancer is breast cancer.
[0432] The compounds described herein can be administered alone or together with one or more additional therapeutic agents. The one or more additional therapeutic agents can be administered just prior to, concurrent with, or shortly after the administration of the compounds described herein. The present disclosure also includes pharmaceutical compositions comprising any of the compounds described herein in combination with one or more additional therapeutic agents, and methods of treatment comprising administering such combinations to subjects in need thereof.
[0433] Suitable additional therapeutic agents include, but are not limited to: an EGFR antagonist (e.g., an anti-EGFR antibody [e.g., cetuximab or panitumumab] or small molecule inhibitor of EGFR [e.g., gefitinib or erlotinib]), an antagonist of another EGFR family member such as Her2/ErbB2, ErbB3 or ErbB4 (e.g., anti-ErbB2 [e.g., trastuzumab or T-DM1 {KADCYLA®}], anti-ErbB3 or anti-ErbB4 antibody or small molecule inhibitor of ErbB2, ErbB3 or ErbB4 activity), an antagonist of EGFRvIII (e.g., an antibody that specifically binds EGFRvIII), a cMET antagonist (e.g., an anti-cMET antibody), an IGF1R antagonist (e.g., an anti-IGF1R antibody), a B-raf inhibitor (e.g., vemurafenib, sorafenib, GDC-0879, PLX- anti-PDGFR-β antibody or small molecule kinase inhibitor such as, e.g., imatinib mesylate or sunitinib malate), a PDGF ligand inhibitor (e.g., anti-PDGF-A, -B, -C, or -D antibody, aptamer, siRNA, etc.), a VEGF antagonist (e.g., a VEGF-Trap such as aflibercept, see, e.g., US 7,087,411 (also referred to herein as a "VEGF-inhibiting fusion protein"), anti-VEGF antibody (e.g., bevacizumab), a small molecule kinase inhibitor of VEGF receptor (e.g., sunitinib, sorafenib or pazopanib)), a DLL4 antagonist (e.g., an anti-DLL4 antibody disclosed in US 2009/0142354 such as REGN421), an Ang2 antagonist (e.g., an anti-Ang2 antibody disclosed in US 2011/0027286 such as H1H685P), a FOLH1 antagonist (e.g., an anti-FOLH1 antibody), a STEAP1 or STEAP2 antagonist (e.g., an anti-STEAP1 antibody or an anti- STEAP2 antibody), a TMPRSS2 antagonist (e.g., an anti-TMPRSS2 antibody), a MSLN antagonist (e.g., an anti-MSLN antibody), a CA9 antagonist (e.g., an anti-CA9 antibody), a uroplakin antagonist (e.g., an anti-uroplakin [e.g., anti-UPK3A] antibody), a MUC16 antagonist (e.g., an anti-MUC16 antibody), a Tn antigen antagonist (e.g., an anti-Tn antibody), a CLEC12A antagonist (e.g., an anti- CLEC12A antibody), a TNFRSF17 antagonist (e.g., an anti-TNFRSF17 antibody), a LGR5 antagonist (e.g., an anti-LGR5 antibody), a monovalent CD20 antagonist (e.g., a monovalent anti-CD20 antibody such as rituximab), etc. Other agents that may be beneficially administered in combination with compounds of the disclosure include, e.g., tamoxifen, aromatase inhibitors, and cytokine inhibitors, including small-molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or to their respective receptors.
[0434] Suitable therapeutic agents also include, but are not limited to
chemotherapeutic agents, including alkylating agents such as thiotepa and
cyclosphosphamide (Cytoxan™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa;
ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5- fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;
mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK™; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethan; vindesine; dacarbazine;
mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g. paclitaxel (Taxol™, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (Taxotere™; Aventis Antony, France);
chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and
pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
[0435] The compounds described herein can also be administered and/or co- formulated in combination with antivirals, antibiotics, analgesics, corticosteroids, steroids, NSAIDs.
[0436] In some embodiments of the methods described herein, multiple doses of a compound described herein (or a pharmaceutical composition comprising a combination of an compound described herein and any of the additional therapeutic agents mentioned herein) may be administered to a subject over a defined time course. The methods according to this aspect of the disclosure comprise sequentially administering to a subject multiple doses of a compound described herein. As used herein, "sequentially administering" means that each dose of the compound is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks or months). The present disclosure includes methods which comprise sequentially administering to the patient a single initial dose of a compound described herein, followed by one or more secondary doses of the compound, and optionally followed by one or more tertiary doses of the compound.
[0437] The terms "initial dose," "secondary doses," and "tertiary doses," refer to the temporal sequence of administration of the compounds described herein. Thus, the "initial dose" is the dose which is administered at the beginning of the treatment regimen (also referred to as the "baseline dose"); the "secondary doses" are the doses which are
administered after the initial dose; and the "tertiary doses" are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses can all contain the same amount the compound described herein, but generally can differ from one another in terms of frequency of administration. In certain embodiments, the amount of the compound contained in the initial, secondary and/or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as "loading doses" followed by subsequent doses that are administered on a less frequent basis (e.g., "maintenance doses").
[0438] In certain exemplary embodiments of the present disclosure, each secondary and/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more) weeks after the immediately preceding dose. The phrase "the immediately preceding dose," as used herein, means, in a sequence of multiple administrations, the dose the compound sequence with no intervening doses.
[0439] The methods according to this aspect of the disclosure may comprise administering to a patient any number of secondary and/or tertiary doses of the compound. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient. The administration regimen may be carried out indefinitely over the lifetime of a particular subject, or until such treatment is no longer therapeutically needed or advantageous.
[0440] In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1 to 2 weeks or 1 to 2 months after the immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2 to 12 weeks after the immediately preceding dose. In certain embodiments of the disclosure, the frequency at which the secondary and/or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.
[0441] The present disclosure includes administration regimens in which 2 to 6 loading doses are administered to a patient at a first frequency (e.g., once a week, once every two weeks, once every three weeks, once a month, once every two months, etc.), followed by administration of two or more maintenance doses to the patient on a less frequent basis. For example, according to this aspect of the disclosure, if the loading doses are administered at a frequency of once a month, then the maintenance doses may be administered to the patient once every six weeks, once every two months, once every three months, etc.
[0442] The present disclosure includes pharmaceutical compositions of the compounds and/or conjugates described herein, e.g., the compounds of Formula (I) and (II), e.g., compositions comprising a compound described herein, a salt, stereoisomer, polymorph thereof, and a pharmaceutically acceptable carrier, diluent, and/or excipient. Examples of suitable carriers, diluents and excipients include, but are not limited to: buffers for buffers, phosphate buffers, lactate buffers, oxalate buffers and the like), carrier proteins (e.g., human serum albumin), saline, polyols (e.g., trehalose, sucrose, xylitol, sorbitol, and the like), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxolate, and the like), antimicrobials, and antioxidants.
F. EXAMPLES [0443] Proton NMR spectra were acquired on Varian Inova 300 or 500 MHz instruments, while mass spectra were collected on an Agilent 1100 or 1200 series LC/MSD with electrospray ionization source and either single-quad or ion trap analyzer. Certain linker payloads in enzymatic assays were analyzed by a Waters Xevo TQ-S mass spectrometer. All starting materials and solvents were purchased commercially and used without purification, unless otherwise noted.
EXAMPLE 1
[0444] Compound 10 was synthesized from Compound 1 as described below and as depicted in FIG 1. Maytansin-N-methyl-L-alanine-4-aminobenzamido-Cit-Val-Cap-Mal (10) [0445] Step A: To a round-bottom flask was weighed Boc-L-valine (1.03 g, 4.74 mmol), N-hydroxysuccinimide (1.22 g, 10.6 mmol), and EDC (1.60 g, 8.35 mmol). The reagents were dissolved in dry DCM (30 mL), the flask sealed via rubber septum, purged with argon, and the reaction stirred at ambient temperature. After 3 days no Boc-valine remained by TLC (after staining with ninhydrin), so the reaction was washed with water and sat. aq. NaHCO3, the aqueous layer extracted with DCM, combined organic layers washed with brine, dried over Na2SO4, and filtered. The filtrate was then evaporated and dried in vacuo giving Boc-L-valine-succinate as a white solid (1.52 g, 100%). 1H-NMR (300 MHz, CDCl3): ^ 4.98 (br d, 1H), 4.58 (dd, 1H), 2.82 (m, 4H), 2.27 (m, 1H), 1.44 (s, 9H), 1.03 (dd, 6H). Boc-L-valine-L-citrulline (3):
[0446] Boc-L-valine-succinate (1) of the preceding step (1.50 g, 4.77 mmol) was dissolved in acetonitrile (MeCN, 15 mL), treated with a solution of L-citrulline (2, 1.07 g, 6.11 mmol) in water (9 mL) and sat. aq. NaHCO3 (6 mL), the flask sealed with a vented 18 h, so additional sat. aq. NaHCO3 (3 mL) was added to bring the pH up to ca.7 and the reaction stirred another 36 h. The reaction was partially concentrated in vacuo to remove MeCN and washed once with ethyl acetate (EtOAc) to remove any nonpolar impurities. The aqueous layer was then acidified to pH 3 with 10% v/v HCl, saturated with NaCl, and extracted 4 times with 9:1 EtOAc/isopropanol. The combined organic extracts were washed with brine, dried over Na2SO4, and filtered. The filtrate was then evaporated and dried in vacuo giving the title compound as a white solid (1.56 g, 87%). MS (ESI, pos.): calc’d for C16H30N4O6, 374.2; found 375.2 (M+H), 397.2 (M+Na). Boc-L-valine-L-citrulline-p-aminobenzoic acid t-butyl ester (5): [0447] Step B: The product of the preceding step (3, 152 mg, 0.406 mmol), tert-butyl- 4-aminobenzoate (4, 150 mg, 0.776 mmol), and 1-[Bis(dimethylamino)methylene]-1H-1,2,3- triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU, 488 mg, 1.28 mmol) were weighed into a round-bottom flask and dissolved in anhydrous N,N-dimethylformamide (DMF, 3 mL). N,N-Diisopropylethylamine (DIEA, 0.25 mL, 1.44 mmol) was added to the reaction, the flask sealed via rubber septum, purged with argon, and the reaction stirred at ambient temperature. After 18 h the reaction was purified directly on a 100g C18 RediSep Gold column via ISCO system (gradient elution: 20– 80% MeCN in water, 0.05% acetic acid in both, over 20 min). The product-containing fractions were combined, partially concentrated in vacuo, frozen on dry ice, and lyophilized overnight giving an impure white solid (115 mg). This was dissolved in DCM and repurified on a 12g silica gel RediSep column via ISCO (gradient elution: 0– 10% methanol in DCM over 12 min), and the slower- running product fractions evaporated and dried in vacuo giving the title compound as a pale yellow solid (65 mg, 29%). MS (ESI, pos.): calc’d for C27H43N5O7, 549.3; found 450.3(M- Boc+H), 572.3 (M+Na), 1099.5 (2M+H), 1121.5 (2M+Na). L-valine-L-citrulline-p-aminobenzoic acid (6): [0448] Step C: The title compound was prepared using the method of Mehta et al. (Tet. Lett.1992, 33, 5441-5444). The product of the preceding step (5, 61 mg, 0.111 mmol) was dissolved in dry DCM (3 mL) in a round-bottom flask, and treated with trifluoroacetic acid (TFA, 110 uL, 1.44 mmol) and triethylsilane [TES (Et3SiH), 50 uL, 0.313 mmol]. The The reaction was incomplete by LCMS, so additional TFA (90 uL) and TES (25 uL) were added and the reaction stirred another 6 h. The reaction was still incomplete so it was capped and stored at -20 oC for 3 d. After warming to ambient temperature and stirring another 24 h it was concentrated in vacuo to an oil, triturated twice with diethyl ether, and dried under high vacuum giving the title compound as an off-white solid (55 mg, 98%). MS (ESI, pos.):
calc’d for C18H27N5O5, 393.2; found 394.0 (M+H), 787.2 (2M+H). 1H-NMR (500 MHz, DMSO-d6) showed a mixture of amide rotamers: ^ 10.52 (s, 0.5H), 10.46 (s, 0.5H), 8.83 (d, 0.5H), 8.71 (d, 0.5H), 8.06 (br s, 3H), 7.89 (m, 2H), 7.72 (m, 2H), 6.03 (m, 1H), 4.55 (m, 1H), 3.05 (m, 1H), 2.97 (m, 1H), 2.10 (m, 1H), 1.73 (m, 1H), 1.63 (m, 1H), 1.5– 1.3 (m, 2H), 0.95 (m, 6H). 6-(Maleimidyl-caprolyl)-L-valine-L-citrulline-p-aminobenzoic acid (8): [0449] Step D: The product of the preceding step (6, 55 mg, 0.108 mmol) was dissolved in water (3 mL), treated with sat. aq. NaHCO3, then with a solution of 6- maleimidyl-caproic acid succinate ester (56 mg, 0.182 mmol) in MeCN (3 mL). The flask was capped under argon and the reaction stirred at ambient temperature for 22 h. The reaction was complete by LCMS, so it was partially concentrated in vacuo and purified directly on a 30g C18 Aq RediSep Gold column via ISCO (gradient elution: 20– 80% MeCN in water, 0.05% acetic acid in both, over 12 min). The major product fractions were combined, partially concentrated in vacuo, frozen on dry ice, and lyophilized overnight giving an impure pale yellow solid (92 mg). This was found to be impure by LCMS so it was dissolved in MeCN/water and repurified on a 100 g C18 Aq Gold column (gradient elution: 0 – 50% MeCN in water, 0.05% acetic acid in both, over 20 min). The cleanest product fractions were combined, partially concentrated in vacuo, frozen on dry ice, and lyophilized giving the title compound as a white solid (34 mg, 53%). MS (ESI, pos.): calc’d for
C28H38N6O8, 586.3; found 587.3 (M+H), 609.3 (M+Na). 1H-NMR (500 MHz, DMSO-d6) showed a mixture of amide rotamers: ^ 10.26 (s, 0.6H), 10.11 (s, 0.4H), 8.43 (d, 0.4H), 8.13 (d, 0.6H), 7.93– 7.70 (m, 4H), 6.99 (m, 2H), 5.97 (m, 1H), 5.41 (m, 2H), 4.38 (m, 1H), 4.21 – 4.12 (m, 1H), 3.36 (m, 2H), 3.02 (m, 1H), 2.95 (m, 1H), 2.17 (m, 1H), 2.12 (m, 1H), 1.95 (m, 1H), 1.78– 1.58 (m, 2H), 1.48 (m, 6H), 1.36 (m, 1H), 1.18 (m, 2H), 0.85 (m, 6H). (10): [0450] Step E: The product of the preceding step (8, 33 mg, 0.056 mmol), HATU (33 mg, 0.087 mmol), and maytansin-N-methyl-L-alanine (9, prepared as a gold solid from maytansinol using the methods described in U.S. Patent Application 2007/0037972 A1, 25 mg, 0.038 mmol), were weighed into a round-bottom flask, dissolved in anhydrous DMF (2 mL), and treated with DIEA (20 uL, 0.115 mmol). The flask was sealed via rubber septum, purged with argon, and the reaction stirred at ambient temperature for 20 h. The reaction was diluted with water (1 mL) and purified directly on a 50g C18 Aq RediSep Gold column via ISCO (gradient elution: 20– 80% MeCN in water, 0.05% acetic acid in both, over 12 min). The product fractions were combined, partially concentrated in vacuo, frozen on dry ice, and lyophilized overnight giving the title compound as a white solid (8 mg, 17%). MS (ESI, pos.): calc’d for C60H80N9O16Cl, 1217.5; found 1218.6 (M+H), 1200.7 (M-H2O+H), 1240.7 (M+Na). 1H-NMR (500 MHz, CDCl3): ^ 9.25 (s, 1H), 7.68– 7.61 (m, 2H), 7.33 (d, 2H), 6.91 (s, 1H), 6.84 (s, 1H), 6.75 (d, 1H), 6.67 (s, 2H), 6.45 (dd, 1H), 6.27 (br s, 1H), 6.21 (d, 1H), 5.74 (dd, 1H), 5.44 (m, 1H), 4.98 (m, 1H), 4.88 (d, 1H), 4.77 (t, 1H), 4.53 (br s, 1H), 4.33– 4.25 (m, 2), 4.00 (s, 3H), 3.65 (d, 1H), 3.48 (m, 4H), 3.56 (s, 3H), 3.20 (m, 1H), 3.11 (d, 1H), 3.05 (m, 3H), 2.88 (s, 3H), 2.69 (t, 1H), 2.26– 2.19 (m, 3H), 2.10 (m, 2H), 1.94 (m, 1H), 1.70– 1.55 (m, 6H), 1.66 (s, 3H), 1.46 (d, 3H), 1.33– 1.26 (m, 7H), 0.96 (m, 6H), 0.85 (s, 3H). EXAMPLE 2
[0451] Compound 15 was synthesized as described below and as depicted in FIG 6. Maytansin-N-methyl-L-alanine-(4-amino-2-fluoro)benzamido-Cit-Val-Cap-Mal (15) Boc-L-valine-L-citrulline-(4-amino-2-fluoro)benzoic acid t-butyl ester (12):
[0452] Step A: Following the procedure of Wipf & Heimgartner (Helv. Chim. Acta, 1998, 71, 140-154), Boc-L-valine-L-citrulline (3, 155 mg, 0.414 mmol) and
dicyclohexylcarbodiimide (DCC, 95 mg, 0.460 mmol) were dissolved in dry dichloromethane (DCM, 3 mL), cooled to 0oC, and stirred for 5 min. (+)-Camphor-10-sulfonic acid (CSA, 15 mg, 0.065 mmol) and tert-butyl-4-amino-2-fluorobenzoate (99 mg, 0.469 mmol) were then added dry and the reaction allowed to slowly warm to ambient temperature while stirring for diluted with DCM and washed with 10% v/v HCl, water, and saturated NaHCO3. The aqueous layers were each extracted once with DCM, and the combined organic layers washed with brine, dried over Na2SO4, and filtered. The filtrate was then evaporated and dried in vacuo giving a pale gold solid which was purified on a 24g RediSep Gold column via ISCO (gradient elution: Ethyl Acetate– 5:5:1 EtOAc/DCM/methanol over 12 min). The cleanest product fractions were combined, concentrated in vacuo, and dried under high vacuum giving the title compound as a white solid (95 mg, 40%). MS (ESI, pos.): calc’d for C27H42N5O7F, 567.3; found 568.3 (M+H), 590.4 (M+Na).
L-valine-L-citrulline-(4-amino-2-fluoro)benzoic acid trifluoroacetate salt (13):
[0453] Step B: The title compound was prepared from the product of the preceding step (12, 94 mg, 0.166), using Step C, Example 1, to give an off-white solid (112 mg). MS (ESI, pos.): calc’d for C18H26N5O5F, 411.2; found 412.2 (M+H), 395.2 (M-H2O+H).
6-(Maleimido)-caproamidyl-L-valine-L-citrulline-(4-amino-2-fluoro)benzoic acid (14):
[0454] Step C: The title compound was prepared from the product of the preceding step (13, 106 mg, 0.166 mmol), using Step D, Example 1, to give a white solid (92 mg) that was only 70% pure by LCMS but used without further purification. MS (ESI, pos.): calc’d for C28H37N6O8F, 604.3; found 605.2 (M+H), 627.2 (M+Na).
Maytansin-N-methyl-L-alanine-(4-amino-2-fluoro)benzamido-Cit-Val-Cap-Mal (15):
[0455] Step D: The title compound was prepared from the product of the preceding step (14, 50 mg, 0.077 mmol) and maytansin-N-methyl-L-alanine (9, 50 mg, 0.077 mmol), using Step E, Example 1, to give a white solid (18 mg) that was only 55% pure by LCMS. Purifying twice by HPLC using a Phenomenex Gemini C185u, 30x150 mm column (20– 80%, then 40– 60%, MeCN in water, 0.1% HOAc both phases, over 20 min, 30 mL/min) gave the title compound as a white solid (3 mg, 3%). MS (ESI, pos.): calc’d for
C60H79N9O16ClF, 1235.5; found 1236.5 (M+H), 1258.5 (M+Na). 1H-NMR (500 MHz, CDCl3): ^ 9.40 (s, 1H), 7.64 (d, 1H, J = 12 Hz), 7.42 (s, 1H), 7.14 (t, 1H, J = 8 Hz), 6.90 (s, 1H), 6.86 (s, 1H), 6.77 (m, 1H), 6.68 (s, 2H), 6.46 (dd, 1H, J = 15 Hz, 11 Hz), 6.25 (br s, Hz), 4.76 (m, 1H), 4.29 (t, 1H, J = 11 Hz), 4.23 (t, 1H, J = 7 Hz), 4.01 (s, 2H), 3.99 (m, 1H), 3.70 (m, 1H), 3.53– 3.47 (m, 4H), 3.36 (s, 3H), 3.20 (m, 1H), 3.13 (d, 1H, J = 12 Hz), 3.03 (m, 3H), 2.81 (s, 2H), 2.67 (dd, 1H, J = 15 Hz, 12 Hz), 2.25 (t, 1H, J = 7 Hz), 2.21 (m, 2H), 2.10 (m, 1H), 1.70– 1.64 (m, 2H), 1.67 (s, 3H), 1.46– 1.41 (m, 6H), 1.33– 1.25 (m, 10H), 0.99– 0.95 (m, 6H), 0.89– 0.80 (m, 1H), 0.85 (s, 3H).
EXAMPLE 3
[0456] Compound 20 was synthesized as described below and as depicted in FIG 7.
Maytansin-N-methyl-L-alanine-(4-amino-2-trifluoromethyl)benzamido-Cit-Val-Cap-Mal (20) Boc-L-valine-L-citrulline-(4-amino-2-trifluoromethyl)benzoic acid t-butyl ester (17):
[0457] Step A: The title compound was prepared from Boc-L-valine-L-citrulline (3, 175 mg, 0.467 mmol) and tert-butyl-4-amino-2-trifluoromethylbenzoate (150 mg, 0.574 mmol), using the method of Wipf & Heimgartner (Helv. Chim. Acta, 1998, 71, 140-154) to give a white solid (77 mg, 27%). MS (ESI, neg.): calc’d for C28H42N5O7F3, 617.3; found 616.4 (M-H).
L-valine-L-citrulline-(4-amino-2-trifluoromethyl)benzoic acid trifluoroacetate salt (18):
[0458] Step B: The title compound was prepared from the product of the preceding step (17, 67 mg, 0.108), using Step C, Example 1, to give an off-white solid (77 mg). MS (ESI, pos.): calc’d for C19H26N5O5F3, 461.2; found 462.3 (M+H), 445.2 (M-H2O+H).
6-(Maleimido)-caproamidyl-L-valine-L-citrulline-(4-amino-2-trifluoromethyl)benzoic acid (19):
[0459] Step C: The title compound was prepared from the product of the preceding step (18, 75 mg, 0.108 mmol), using Step D, Example 1, to give a white solid (47 mg, 66%). MS (ESI, pos.): calc’d for C29H37N6O8F3, 654.3; found 655.3 (M+H).
Figure imgf000238_0001
(20):
[0460] Step D: The title compound was prepared from the product of the preceding step (19, 34 mg, 0.052 mmol) and maytansin-N-methyl-L-alanine (9, 34 mg, 0.052 mmol), using Step E, Example 1, to give a white solid (11 mg, 16%) after a second ISCO purification (100g C18 Aq Gold column, 30– 70% MeCN in water, 0.05% HOAc both, over 15 min, 50 mL/min). MS (ESI, pos.): calc’d for C61H79N9O16ClF3, 1285.5; found 1287.4 (M+H), 1268.4 (M-H2O+H), 1308.4 (M+Na). 1H-NMR (500 MHz, DMSO-d6): ^ 10.4 (s, 1H), 8.16 (d, 1H, J = 7 Hz), 8.13 (s, 1H), 7.80 (d, 1H, J = 8 Hz), 7.25 (s, 1H), 7.12 (m, 1H), 6.99 (s, 2H), 6.93 (s, 1H), 6.84 (s, 1H), 6.63– 6.55 (m, 2H), 6.01 (s, 1H), 5.95 (m, 1H), 5.58 (dd, 1H, J = 15 Hz, 9 Hz), 5.38 (m, 3H), 4.64 (dd, 1H, J = 12 Hz, 3 Hz), 4.30 (m, 1H), 4.17– 4.08 (m, 2H), 3.96 (s, 2H), 3.93 (m, 1H), 3.53 (d, 1H, J = 9 Hz), 3.40 (br m, 1H), 3.36 (m, 1H), 3.27 (s, 3H), 3.25 (m, 1H), 3.04 (s, 3H), 3.03– 2.92 (m, 2H), 2.84– 2.70 (m, 2H), 2.53 (m, 2H), 2.20– 2.09 (m, 3H), 1.94 (m, 1H), 1.70 (m, 1H), 1.64 (s, 3H), 1.60– 1.53 (m, 3H), 1.51– 1.44 (m, 6H), 1.40 (m, 1H), 1.37– 1.32 (m, 3H), 1.30– 1.24 (m, 2H), 1.20– 1.12 (m, 5H), 0.89 (m, 1H), 0.86 (s, 3H), 0.84– 0.80 (m, 6H).
EXAMPLE 4
[0461] Compound 25 was synthesized as described below and as depicted in FIG 8.
Maytansin-N-methyl-L-alanine-(4-amino-2-methoxy)benzamido-Cit-Val-Cap-Mal (25) Boc-L-valine-L-citrulline-(4-amino-2-methoxy)benzoic acid t-butyl ester (22):
[0462] Step A: The title compound was prepared from Boc-L-valine-L-citrulline (3, 143 mg, 0.382 mmol) and tert-butyl-4-amino-2-methoxybenzoate (109 mg, 0.488 mmol), using the method of Wipf & Heimgartner (Helv. Chim. Acta, 1998, 71, 140-154) to give a white solid (92 mg, 42%). MS (ESI, neg.): calc’d for C28H45N5O8, 579.3; found 580.3 (M- H), 602.3 (M+Na).
L-valine-L-citrulline-(4-amino-2-methoxy)benzoic acid trifluoroacetate salt (23):
[0463] Step B: The title compound was prepared from the product of the preceding step (22, 90 mg, 0.155), using Step C, Example 1, to give a pale solid (99 mg) that was in vacuo to give the title compound as an off-white solid (79 mg, 95%). MS (ESI, pos.): calc’d for C19H29N5O6, 423.2; found 424.2 (M+H), 407.2 (M-H2O+H), 446.2 (M+Na).
6-(Maleimido)-caproamidyl-L-valine-L-citrulline-(4-amino-2-methoxy)benzoic acid (24):
[0464] Step C: The title compound was prepared from the product of the preceding step (23, 76 mg, 0.141 mmol), using Step D, Example 1, to give a white solid (50 mg, 57%). MS (ESI, pos.): calc’d for C29H40N6O9, 616.3; found 617.2 (M+H).
Maytansin-N-methyl-L-alanine-(4-amino-2-methoxy)benzamido-Cit-Val-Cap-Mal (25):
[0465] Step D: The title compound was prepared from the product of the preceding step (24, 49 mg, 0.079 mmol) and maytansin-N-methyl-L-alanine (9, 34 mg, 0.052 mmol), using Step E, Example 1, to give a white solid (34 mg, 34%). MS (ESI, pos.): calc’d for C61H82N9O17Cl, 1247.6; found 1248.5 (M+H), 1230.5 (M-H2O+H), 1270.5 (M+Na). 1H- NMR (500 MHz, DMSO-d6): ^ 9.26 (br s, 1H), 7.49 (br m, 1H), 6.99 (m, 1H), 6.95 (s, 1H), 6.87 (s, 1H), 6.85 (m, 1H), 6.69 (s, 2H), 6.45 (dd, 1H, J = 15 Hz, 11 Hz), 6.29 (br s, 1H), 5.73 (dd, 1H, J = 16 Hz, 10 Hz), 4.84 (d, 1H, J = 12 Hz), 4.70 (br m, 1H), 4.30 (t, 1H, J = 12 Hz), 4.01 (s, 3H), 3.74 (br m, 4H), 3.55– 3.47 (m, 5H), 3.35 (s, 3H), 3.14 (m, 2H), 3.02 (m, 4H), 2.83 (br s, 3H), 2.71 (s, 3H), 2.65 (m, 1H), 2.60 (t, 1H, J = 8 Hz), 2.24– 2.18 (m, 3H), 2.09 (m, 1H), 1.77 (pentet, 1H, J = 8 Hz), 1.70– 1.61 (m, 6H), 1.67 (s, 3H), 1.51– 1.40 (m, 6H), 1.30– 1.26 (m, 5H), 0.94 (m, 6H), 0.85 (s, 3H).
EXAMPLE 5
[0466] Compound 27 was synthesized from Compound 26 as described below and as depicted in FIG 9. Maytansin-N-methyl-L-alanine-4-aminobenzamide (27) Step A: Maytansin-N-methyl-L-alanine-(4-nitro)benzamide:
[0467] To a dry, round-bottom flask was weighed maytansin-N-methyl-L-alanine (9, 96 mg, 0.15 mmol), 4-nitrobenzoic acid (26) (42 mg, 0.25 mmol), and HATU (0.12 g, 0.31 mmol). The reagents were dissolved in anhydrous DMF (3.0 mL), treated with DIEA (0.10 reaction was stirred at ambient temperature for 3 d, after which LCMS showed complete conversion of Maytan-NMA, so it was diluted with a few mL of water and purified directly on a 100 g C18 Aq Gold column (gradient elution: 20– 80% MeCN in water, 0.05% acetic acid in both, over 15 min). The cleanest product fractions were combined, partially concentrated in vacuo, frozen on dry ice, and lyophilized overnight giving the title compound as a yellow solid (64 mg, 54%). MS (ESI, pos.): calc’d for C39H47N4O12Cl, 798.3; found 798.4 (M+H). Step B: Maytan-NMA-(4-amino)benzamide (27):
[0468] The product of the preceding step (63 mg, 0.079 mmol) and zinc dust (<10 um, 98+% pure, 108 mg, 1.65 mmol) were dissolved/suspended in a mixture of THF (4 mL) and water (1 mL). Acetic acid (0.180 mL, 3.14 mmol) was added to the mixture, the flask sealed with a rubber septum, and the reaction stirred at ambient temperature for 1 h. LCMS of the crude mixture showed complete conversion, so the reaction was filtered over Celite, washed off with MeCN, and the filtrate concentrated in vacuo. The crude product was purified directly on a 50 g C18 Aq Gold column (gradient elution: 20– 80% MeCN in water, 0.05% acetic acid in both, over 12 min). The cleanest product fractions were combined, partially concentrated in vacuo, frozen on dry ice, and lyophilized overnight giving 46 mg of white solid that was only 88% pure by LCMS. This was dissolved in 1:1 MeCN/water (3 mL) and repurified by HPLC using a Phenomenex Gemini C185u, 30x150 mm column in two injections (40– 80% and 30– 70% MeCN in water, 0.05% HOAc both phases, over 20 min, 30 mL/min), and the cleanest fraction were concentrated, frozen, and lyophilized as above giving the title compound as a white solid (31 mg, 48%). MS (ESI, pos.): calc’d for
C41H53N4O12Cl, 768.3; found 751.2 (M-H2O+H), 769.2 (M+H). 1H-NMR (500 MHz, CDCl3): ^ 7.24 (d, 2H, J = 9 Hz), 6.93 (s, 1H), 6.82 (s, 1H), 6.76 (d, 1H, J = 12 Hz), 6.57 (d, 2H, J = 9 Hz), 6.45 (dd, 1H, J = 16 Hz, 12 Hz), 6.23 (s, 1H), 5.74 (dd, 1H, J = 16 Hz, 9 Hz), 5.43 (br m, 1H), 4.87 (dd, 1H, J = 12 Hz, 3 Hz), 4.32 (m, 1H), 3.99 (s, 3H), 3.85 (s, 2H), 3.65 (d, 1H, J = 13 Hz), 3.51 (d, 1H, J = 9 Hz), 3.47 (br s, 1H), 3.36 (s, 3H), 3.10 (d, 1H, J = 13 Hz), 3.07 (s, 3H), 3.04 (d, 1H, J = 9 Hz), 2.93 (s, 3H), 2.67 (m, 1H), 2.20 (dd, 1H, J = 14 Hz, 3 Hz), 1.67 (m, 1H), 1.66 (s, 3H), 1.51– 1.47 (m, 2H), 1.44 (d, 3H, J = 7 Hz), 1.31 (d, 3H, J = 7 Hz), 1.27 (m, 1H), 0.84 (s, 3H). [0469] Compound 29 was synthesized from Compound 28 as described below and as depicted in FIG 10. Maytansin-N-methyl-L-alanine-(2-fluoro-4-amino)benzamide (29) Step A: Maytansin-N-methyl-L-alanine-(2-fluoro-4-nitro)benzamide:
[0470] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 40 mg, 0.056 mmol) and 2-fluoro-4-nitrobenzoic acid (28) (26 mg, 0.140 mmol), using Step A of Example 5, to give a light yellow solid (16 mg, 35%). MS (ESI, pos.): calc’d for
C39H46N4O12ClF, 816.3; found 817.2 (M+H), 839.2 (M+Na). Step B: Maytan-NMA-(2-fluoro-4-amino)benzamide (29):
[0471] The title compound was prepared from the product of the preceding step (15 mg, 0.018 mmol), using Step B of Example 5, to give a white solid (8 mg, 50%). MS (ESI, pos.): calc’d for C39H48N4O10ClF, 786.3; found 769.2 (M-H2O+H), 787.2 (M+H), 809.3 (M+Na). 1H-NMR (500 MHz, CDCl3): ^ 7.05– 6.99 (m, 2H), 6.92 (s, 1H), 6.85 (s, 1H), 6.81 (d, 1H, J = 11 Hz), 6.47 (dd, 1H, J = 15 Hz, 11 Hz), 6.36– 6.29 (m, 2H), 6.22 (s, 1H), 5.73 (dd, 1H, J = 16 Hz, 9 Hz), 5.48 (m, 1H), 4.87 (dd, 1H, J = 12 Hz, 3 Hz), 4.30 (m, 1H), 4.00 (s, 3H), 3.93 (s, 2H), 3.73 (d, 1H, J = 13 Hz), 3.51 (d, 1H, J = 9 Hz), 3.41 (br m, 1H), 3.36 (s, 3H), 3.13 (d, 1H, J = 12 Hz), 3.04 (s, 3H), 3.02 (m, 1H), 2.83 (s, 3H), 2.66 (dd, 1H, J = 15 Hz, 13 Hz), 2.19 (dd, 1H, J = 15 Hz, 3 Hz), 1.67 (s, 3H), 1.63 (m, 1H), 1.51– 1.45 (m, 2H), 1.43 (d, 3H, J = 7 Hz), 1.30 (d, 3H, J = 7 Hz), 1.27 (m, 1H), 0.84 (s, 3H).
EXAMPLE 7
[0472] Compound 31 was synthesized as described below and as depicted in FIG 11. Maytansin-N-methyl-L-alanine-(2-trifluoromethyl-4-amino)benzamide (31) Step A: Maytansin-N-methyl-L-alanine-(2-trifluoromethyl-4-nitro)benzamide:
[0473] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 68 mg, 0.105 mmol) and 2-trifluoromethyl-4-nitrobenzoic acid (30) (37 mg, 0.157 mmol), using Step A of Example 5, to give a pale yellow solid (82 mg, 90%). MS (ESI, pos.): calc’d for C40H46N4O12ClF3, 866.3; found 867.1 (M+H), 889.1 (M+Na). [0474] The title compound was prepared from the product of the preceding step (79 mg, 0.091 mmol), using Step B of Example 5, to give a white solid (29 mg, 35%). MS (ESI, pos.): calc’d for C40H48N4O10ClF3, 836.3; found 818.8 (M-H2O+H), 836.8 (M+H), 858.0 (M+Na). 1H-NMR (500 MHz, CDCl3): ^ 7.00– 6.91 (m, 3H), 6.85 (d, 1H, J = 3 Hz), 6.75 (br d, 1H, J = 18 Hz), 6.63 (d, 1H, J = 11 Hz), 6.45 (dd, 1H, J = 26 Hz, 19 Hz), 6.23 (s, 1H), 5.73 (dd, 1H, J = 26 Hz, 15 Hz), 4.88 (dd, 1H, J = 20 Hz, 5 Hz), 4.31 (m, 1H), 4.01 (s, 3H), 3.96 (m, 1H), 3.66 (d, 1H, J = 22 Hz), 3.52 (d, 1H, J = 15 Hz), 3.37 (s, 3H), 3.14 (s, 3H), 3.11 (m, 1H), 3.03 (d, 1H, J = 16 Hz), 2.72 (m, 1H), 2.66 (s, 3H), 2.23 (dd, 1H, J = 24 Hz, 5 Hz), 1.66 (s, 3H), 1.51– 1.45 (m, 2H), 1.43 (d, 3H, J = 12 Hz), 1.31 (d, 3H, J = 11 Hz), 1.27 (m, 1H), 0.87 (s, 3H).
EXAMPLE 8
[0475] Compound 33 was synthesized as described below and as depicted in FIG 12.
Maytansin-N-methyl-L-alanine-(2-methoxy-4-amino)benzamide (33) Step A: Maytansin-N-methyl-L-alanine-(2-methoxy-4-nitro)benzamide:
[0476] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 68 mg, 0.105 mmol) and 2-methoxy-4-nitrobenzoic acid (32) (32 mg, 0.162 mmol), using Step A of Example 5, to give a pale yellow solid (78 mg, 90%). MS (ESI, pos.): calc’d for C40H49N4O13Cl, 828.3; found 811.1 (M-H2O+H), 829.2 (M+H), 851.2 (M+Na). Step B: Maytan-NMA-(2-methoxy-4-amino)benzamide (33):
[0477] The title compound was prepared from the product of the preceding step (75 mg, 0.090 mmol), using Step B of Example 5, to give a white solid (62 mg, 79%). MS (ESI, pos.): calc’d for C40H51N4O11Cl, 798.3; found 781.2 (M-H2O+H), 799.2 (M+H), 821.2 (M+Na). 1H-NMR (500 MHz, CDCl3): ^ 7.03 (d, 1H, J = 14 Hz), 6.99 (s, 1H), 6.94 (d, 1H, J = 12 Hz), 6.86 (s, 1H), 6.81 (d, 1H, J = 8 Hz), 6.46 (dd, 1H, J = 16 Hz, 11 Hz), 6.24 (s, 1H), 6.17 (s, 1H), 6.11 (d, 1H, J = 8 Hz), 5.73 (dd, 1H, J = 15 Hz, 9 Hz), 5.54 (m, 1H), 4.81 (m, 1H), 4.31 (t, 1H, J = 11 Hz), 4.00 (s, 3H), 3.81 (d, 1H, J = 13 Hz), 3.79 (m, 1H), 3.52 (d, 1H, J = 9 Hz), 3.36 (s, 3H), 3.12 (d, 1H, J = 13 Hz), 3.04 (m, 1H), 3.02 (s, 3H), 2.72 (s, 3H), 2.64 2H), 1.41 (d, 3H, J = 7 Hz), 1.30 (d, 3H, J = 7 Hz), 1.26 (m, 1H), 0.84 (s, 3H). EXAMPLE 9
[0478] Compound 35 was synthesized as described below and as depicted in FIG 13. Maytansin-N-methyl-L-alanine-N-(3-trifluoromethyl-4-amino)benzamide (35) Step A: Maytansin-N-methyl-L-alanine-(3-trifluoromethyl-4-nitro)benzamide:
[0479] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 46 mg, 0.071 mmol) and 3-trifluoromethyl-4-nitrobenzoic acid (34) (25 mg, 0.106 mmol), using the method from Step A of Example 5, to give a light yellow solid (37 mg, 61%). MS (ESI, pos.): calc’d for C40H46N4O12ClF3, 866.3; found 849.2 (M-H2O+H), 867.2 (M+H), 889.2 (M+Na). Step B: Maytansin-N-methyl-L-alanine-N-(3-trifluoromethyl-4-amino)benzamide (35):
[0480] The title compound was prepared from the product of the preceding step (36 mg, 0.042 mmol), using the method from Step B of Example 5, to give a white solid (17 mg, 46%). MS (ESI, pos.): calc’d for C40H48N4O10ClF3, 836.3; found 819.2 (M-H2O+H), 837.2 (M+H), 859.2 (M+Na). 1H-NMR (500 MHz, CDCl3): d 7.54 (s, 1H), 7.37 (d, 1H, J = 8 Hz), 6.91 (br s, 1H), 6.83 (s, 1H), 6.70 (d, 1H, J = 11 Hz), 6.66 (d, 1H, J = 8 Hz), 6.45 (dd, 1H, J = 15 Hz, 11 Hz), 6.28 (s, 1H), 5.73 (dd, 1H, J = 16 Hz, 9 Hz), 5.44 (m, 1H), 4.88 (dd, 1H, J = 12 Hz, 3 Hz), 4.40 (s, 2H), 4.30 (t, 1H, J = 11 Hz), 3.99 (s, 3H), 3.63 (d, 1H, J = 13 Hz), 3.52 (d, 1H, J = 9 Hz), 3.36 (s, 3H), 3.12 (d, 1H, J = 13 Hz), 3.03 (m, 4H), 2.92 (s, 3H), 2.69 (m, 1H), 2.21 (dd, 1H, J = 14 Hz, 3 Hz), 1.65 (m, 4H), 1.52– 1.44 (m, 4H), 1.31 (d, 3H, J = 6 Hz), 1.26 (m, 2H), 0.85 (s, 3H). Example 10
[0481] Compound 37 was synthesized as described below and as depicted in FIG 14.
Maytansin-N-methyl-L-alanine-N-(2-chloro-4-amino-5-fluoro)benzamide (37) Step A: Maytansin-N-methyl-L-alanine-(2-chloro-4-nitro-5-fluoro)benzamide: mg, 0.071 mmol) and 3-trifluoromethyl-4-nitrobenzoic acid (36,26 mg, 0.118 mmol), using the method from Step A of Example 5, to give a white solid (33 mg, 55%). MS (ESI, pos.): calc’d for C39H45N4O12Cl2F, 850.2; found 833.1 (M-H2O+H), 851.1 (M+H), 873.1 (M+Na). Step B: Maytansin-N-methyl-L-alanine-N-(2-chloro-4-amino-5-fluoro)benzamide (37):
[0483] The title compound was prepared from the product of the preceding step (36 mg, 0.042 mmol), using the method from Step B of Example 5, to give a white solid (17 mg, 46%). MS (ESI, pos.): calc’d for C39H47N4O10Cl2F, 820.3; found 803.2 (M-H2O+H), 821.2 (M+H), 843.2 (M+Na). 1H-NMR (500 MHz, CDCl3): d 6.88 (d, 2H, J = 13 Hz), 6.83 (d, 1H, J = 11 Hz), 6.77 (d, 1H, J = 8 Hz), 6.72 (d, 1H, J = 10 Hz), 6.46 (dd, 1H, J = 15 Hz, 11 Hz), 6.25 (s, 1H), 5.71 (dd, 1H, J = 16 Hz, 10 Hz), 5.52 (m, 1H), 4.85 (dd, 1H, J = 12 Hz, 3 Hz), 4.30 (t, 1H, J = 11 Hz), 4.01 (s, 3H), 3.92 (s, 2H), 3.73 (d, 1H, J = 13 Hz), 3.52 (d, 1H, J = 9 Hz), 3.37 (s, 3H), 3.15 (d, 1H, J = 13 Hz), 3.10 (s, 3H), 3.03 (d, 1H, J = 10 Hz), 2.75 (s, 3H), 2.68 (dd, 1H, J = 14 Hz, 12 Hz), 2.22 (dd, 1H, J = 15 Hz, 3 Hz), 1.67 (s, 3H), 1.61 (m, 1H), 1.50– 1.43 (m, 5H), 1.31 (d, 3H, J = 6 Hz), 1.27– 1.24 (m, 1H), 0.86 (s, 3H). Example 11
[0484] Compound 39 was synthesized as described below and as depicted in FIG 16.
Maytansin-N-methyl-L-alanine-N-(2,5-difluoro-4-amino)benzamide (39) Step A: Maytansin-N-methyl-L-alanine-(2,5-difluoro-4-nitro)benzamide:
[0485] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 48 mg, 0.074 mmol) and 2,5-difluoro-4-nitrobenzoic acid (38, 27 mg, 0.133 mmol), using the method from Step A of Example 5, to give a yellow solid (37 mg, 60%). MS (ESI, pos.): calc’d for C39H45N4O12ClF2, 834.3; found 817.2 (M-H2O+H), 835.2 (M+H), 857.2 (M+Na). Step B: Maytansin-N-methyl-L-alanine-(2,5-difluoro-4-amino)benzamide (39):
[0486] The title compound was prepared from the product of the preceding step (36 mg, 0.043 mmol), using the method from Step B of Example 5, to give a white solid (22 mg, 59%). MS (ESI, pos.): calc’d for C39H47N4O10ClF2, 804.3; found 787.3 (M-H2O+H), 805.3 (M+H), 827.3 (M+Na). 1H-NMR (500 MHz, CDCl3): ^ 6.89– 6.84 (m, 3H), 6.77 (d, 1H, J = 11 Hz), 6.49– 6.41 (m, 2H), 6.24 (s, 1H), 5.72 (dd, 1H, J = 16 Hz, 9 Hz), 5.47 (q, 1H, J = 7 1H, J = 13 Hz), 3.52 (d, 1H, J = 9 Hz), 3.36 (s, 3H), 3.33 (br s, 1H), 3.13 (d, 1H, J = 13 Hz), 3.06 (s, 3H), 3.02 (d, 1H, J = 10 Hz), 2.83 (d, 3H, J = 2 Hz), 2.66 (dd, 1H, J = 15 Hz, 12 Hz), 2.18 (dd, 1H, J = 14 Hz, 3 Hz), 1.67 (s, 3H), 1.63 (d, 1H, J = 14 Hz), 1.51– 1.45 (m, 1H), 1.43 (d, 3H, J = 7 Hz), 1.31 (d, 3H, J = 6 Hz), 1.27 (m, 1H), 0.84 (s, 3H).
Example 12
[0487] Compound 41 was synthesized as described below and as depicted in FIG 17.
Maytansin-N-methyl-L-alanine-(3-fluoro-4-amino)benzamide (41) Step A: Maytansin-N-methyl-L-alanine-(3-fluoro-4-nitro)benzamide:
[0488] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 47 mg, 0.072 mmol) and 3-fluoro-4-nitrobenzoic acid (40) (24 mg, 0.130 mmol), using the method from Step A of Example 5, to give a yellow solid (40 mg, 68%). MS (ESI, pos.): calc’d for C39H46N4O12ClF, 816.3; found 799.2 (M-H2O+H), 817.2 (M+H), 839.2 (M+Na). Step B: Maytansin-N-methyl-L-alanine-(3-fluoro-4-amino)benzamide (41):
[0489] The title compound was prepared from the product of the preceding step (39 mg, 0.048 mmol), using the method from Step B of Example 5, to give a white solid (24 mg, 60%). MS (ESI, pos.): calc’d for C39H48N4O10ClF, 786.3; found 769.2 (M-H2O+H), 787.2 (M+H), 809.2 (M+Na). 1H-NMR (500 MHz, CDCl3): ^ 7.11– 7.04 (m, 2H), 6.89 (s, 1H), 6.83 (d, 1H, J = 2 Hz), 6.72 (d, 1H, J = 11 Hz), 6.68 (t, 1H, J = 8 Hz), 6.45 (dd, 1H, J = 15 Hz, 11 Hz), 6.25 (s, 1H), 5.72 (dd, 1H, J = 15 Hz, 9 Hz), 5.43 (m, 1H), 4.86 (dd, 1H, J = 12 Hz, 3 Hz), 4.31 (t, 1H, J = 11 Hz), 3.99 (s, 3H), 3.92 (m, 2H), 3.62 (d, 1H, J = 13 Hz), 3.51 (d, 1H, J = 9 Hz), 3.40 (bs s, 1H), 3.36 (s, 3H), 3.12 (m, 1H), 3.08 (s, 3H), 3.04 (d, 1H, J = 10 Hz), 2.92 (s, 3H), 2.68 (t, 1H, J = 13 Hz), 2.20 (dd, 1H, J = 15 Hz, 3 Hz), 1.66 (s, 3H), 1.63 (m, 1H), 1.49– 1.46 (m, 1H), 1.44 (d, 3H, J = 7 Hz), 1.31 (d, 3H, J = 7 Hz), 1.27 (m, 1H), 0.84 (s, 3H). Example 13
[0490] Compound 43 was synthesized as described below and as depicted in FIG 18. Maytansin-N-methyl-L-alanine-(3-chloro-4-amino)benzamide (43) Step A: Maytansin-N-methyl-L-alanine-(3-chloro-4-amino)benzamide:
[0491] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 45 mg, 0.069 mmol) and 3-chloro-4-nitrobenzoic acid (42) (26 mg, 0.129 mmol), using the method from Step A of Example 5, to give a yellow solid (36 mg, 62%). MS (ESI, pos.): calc’d for C39H46N4O12Cl2, 832.2; found 815.2 (M-H2O+H), 833.2 (M+H), 855.2 (M+Na). Step B: Maytansin-N-methyl-L-alanine-(3-chloro-4-amino)benzamide (43):
[0492] The title compound was prepared from the product of the preceding step (35 mg, 0.042 mmol), using the method from Step B of Example 5, to give a white solid (24 mg, 67%). MS (ESI, pos.): calc’d for C39H48N4O10Cl2, 802.3; found 785.2 (M-H2O+H), 803.2 (M+H), 825.1 (M+Na). 1H-NMR (500 MHz, CDCl3): ^ 7.35 (s, 1H), 7.17 (d, 1H, J = 8 Hz), 6.90 (s, 1H), 6.83 (d, 1H, J = 2 Hz), 6.68 (m, 2H), 6.44 (dd, 1H, J = 15 Hz, 11 Hz), 6.30 (s, 1H), 5.73 (dd, 1H, J = 15 Hz, 9 Hz), 5.42 (m, 1H), 4.87 (dd, 1H, J = 12 Hz, 3 Hz), 4.32– 4.27 (m, 3H), 3.99 (s, 3H), 3.60 (d, 1H, J = 13 Hz), 3.51 (d, 1H, J = 10 Hz), 3.44 (bs s, 1H), 3.36 (s, 3H), 3.10 (m, 4H), 3.03 (d, 1H, J = 10 Hz), 2.92 (s, 3H), 2.69 (m, 1H), 2.21 (dd, 1H, J = 15 Hz, 3 Hz), 1.65 (s, 3H), 1.63 (m, 1H), 1.52– 1.45 (m, 1H), 1.43 (d, 3H, J = 7 Hz), 1.31 (d, 3H, J = 7 Hz), 1.27 (m, 1H), 0.83 (s, 3H). Example 14
[0493] Compound 45 was synthesized as described below and as depicted in FIG 19.
Maytansin-N-methyl-L-alanine-(5-amino-8-carboxyquinoline)carboxamide (45) Step A: Maytansin-N-methyl-L-alanine-(5-nitro-8-carboxyquinoline)carboxamide :
[0494] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 45 mg, 0.069 mmol) and 5-nitro-8-carboxyquinoline (44) (24 mg, 0.110 mmol), using the method from Step A of Example 5, to give a pale yellow solid (26 mg, 44%). MS (ESI, pos.): calc’d for C42H48N5O12Cl, 849.3; found 832.2 (M-H2O+H), 850.2 (M+H), 872.2 (M+Na). Step B: Maytansin-N-methyl-L-alanine-(5-amino-8-carboxyquinoline)carboxamide (45): mg, 0.029 mmol), using the method from Step B of Example 5, to give a pale yellow solid (8 mg, 31%). MS (ESI, pos.): calc’d for C42H50N5O10Cl, 819.3; found 802.3 (M-H2O+H), 820.3 (M+H). 1H-NMR (500 MHz, DMSO-d6): d 8.80 (br s, 1H), 8.54 (d, 1H, J = 10 Hz), 7.39 (br s, 1H), 7.28 (s, 1H), 7.05– 7.00 (br m, 2H), 6.90 (s, 1H), 6.84 (m, 1H), 6.62 (dd, 1H, J = 15 Hz, 11 Hz), 6.52 (br m, 1H), 6.22 (s, 2H), 5.98 (s, 1H), 5.62 (br m, 2H), 4.56 (br m, 1H), 4.11 (m, 1H), 3.98 (s, 3H), 3.64 (d, 1H, J = 13 Hz), 3.53 (d, 1H, J = 9 Hz), 3.27 (s, 3H), 2.92– 2.88 (m, 3H), 2.81 (d, 1H, J = 10 Hz), 2.42 (br s, 2H), 2.07– 2.04 (m, 1H), 1.75 (m, 2H), 1.66 (s, 3H), 1.55– 1.45 (m, 3H), 1.42 (d, 3H, J = 7 Hz), 1.32 (d, 1H, J = 14 Hz), 1.24 (s, 1H), 1.14 (d, 3H, J = 7 Hz), 0.84 (s, 3H). Example 15
[0496] Compound 47 was synthesized as described below and as depicted in FIG 20.
Maytansin-N-methyl-L-alanine-(3-bromo-4-amino)benzamide (47) Step A: Maytansin-N-methyl-L-alanine-(3-bromo-4-nitro)benzamide :
[0497] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 49 mg, 0.075 mmol) and 3-bromo-4-nitrobenzoic acid (46) (30 mg, 0.122 mmol), using the method from Step A of Example 5, to give a yellow solid (38 mg, 58%). MS (ESI, pos.): calc’d for C39H46N4O12BrCl, 876.2/878.2; found 861.1 (M-H2O+H), 879.1 (M+H), 901.1 (M+Na) for the most abundant isotopes. Step B: Maytansin-N-methyl-L-alanine-(3-bromo-4-amino)benzamide (47):
[0498] The title compound was prepared from the product of the preceding step (37 mg, 0.042 mmol), using the method from Step B of Example 5, to give a white solid (28 mg, 74%). MS (ESI, pos.): calc’d for C39H48N4O10BrCl, 846.2/848.2; found 831.1 (M-H2O+H), 849.1 (M+H), 871.2 (M+Na) for t isotopes. 1H-NMR (500 MHz, CDCl3):
Figure imgf000248_0001
d 7.51 (s, 1H), 7.20 (d, 1H, J = 8 Hz), 6.90 (s, 1H), 6.83 (s, 1H), 6.68 (m, 2H), 6.44 (dd, 1H, J = 15 Hz, 11 Hz), 6.33 (s, 1H), 5.74 (dd, 1H, J = 15 Hz, 10 Hz), 5.42 (m, 1H), 4.88 (dd, 1H, J = 12 Hz, 3 Hz), 4.31 (m, 3H), 3.99 (s, 3H), 3.60 (d, 1H, J = 13 Hz), 3.51 (d, 1H, J = 9 Hz), 3.46 (br s, 1H), 3.36 (s, 3H), 3.10 (s, 3H), 3.09 (m, 1H), 3.03 (d, 1H, J = 10 Hz), 2.70 (t, 1H, 7 Hz), 1.30 (d, 3H, J = 6 Hz), 1.27 (m, 1H), 0.85 (s, 3H). Example 16
[0499] Compound 49 was synthesized as described below and as depicted in FIG 21.
Maytansin-N-methyl-L-alanine-(3-methoxy-4-amino)benzamide (49) Step A: Maytansin-N-methyl-L-alanine-(3-methoxy-4-nitro)benzamide :
[0500] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 49 mg, 0.075 mmol) and 3-methoxy-4-nitrobenzoic acid (48) (23 mg, 0.117 mmol), using the method from Step A of Example 5, to give a yellow solid (34 mg, 55%). MS (ESI, pos.): calc’d for C40H49N4O13Cl, 828.3; found 811.2 (M-H2O+H), 829.3 (M+H), 851.3 (M+Na). Step B: Maytansin-N-methyl-L-alanine-(3-methoxy-4-amino)benzamide (49):
[0501] The title compound was prepared from the product of the preceding step (33 mg, 0.040 mmol), using the method from Step B of Example 5, to give a white solid (25 mg, 74%). MS (ESI, pos.): calc’d for C40H51N4O11Cl, 798.3; found 781.2 (M-H2O+H), 799.2 (M+H). 1H-NMR (500 MHz, CDCl3): d 6.94 (s, 1H), 6.92 (s, 1H), 6.85 (d, 1H, J = 8 Hz), 6.81 (s, 1H), 6.74 (br d, 1H, J = 10 Hz), 6.54 (d, 1H, J = 10 Hz), 6.44 (dd, 1H, J = 15 Hz, 11 Hz), 6.33 (s, 1H), 5.76 (m, 1H), 5.43 (br s, 1H), 4.87 (dd, 1H, J = 12 Hz, 3 Hz), 4.31 (t, 1H, J = 11 Hz), 3.98 (s, 3H), 3.70 (s, 3H), 3.64 (br d, 1H, J = 13 Hz), 3.36 (s, 3H), 3.11– 3.02 (m, 5H), 2.94 (s, 3H), 2.68 (m, 1H), 2.20 (dd, 1H, J = 14 Hz, 3 Hz), 1.67 (m, 1H), 1.65 (s, 3H), 1.49 (dd, 1H, J = 9 Hz, 7 Hz), 1.44 (d, 3H, J = 7 Hz), 1.30 (d, 3H, J = 7 Hz), 1.27 (m, 1H), 0.85 (s, 3H). Example 17
[0502] Compound 51 was synthesized as described below and as depicted in FIG 22.
Maytansin-N-methyl-L-alanine-(2-methyl-4-amino)benzamide (51) Step A: Maytansin-N-methyl-L-alanine-(2-methyl-4-nitro)benzamide : mg, 0.075 mmol) and 2-methyl-4-nitrobenzoic acid (50) (24 mg, 0.132 mmol), using the method from Step A of Example 5, to give a pale yellow solid (32 mg, 52%). MS (ESI, pos.): calc’d for C40H49N4O12Cl, 812.3; found 795.3 (M-H2O+H), 813.3 (M+H), 835.3 (M+Na). Step B: Maytansin-N-methyl-L-alanine-(2-methyl-4-amino)benzamide (51):
[0504] The title compound was prepared from the product of the preceding step (30 mg, 0.037 mmol), using the method from Step B of Example 5, to give a white solid (17 mg, 55%). MS (ESI, pos.): calc’d for C40H51N4O10Cl, 782.3; found 765.2 (M-H2O+H), 783.2 (M+H). 1H-NMR (500 MHz, CDCl3): d 6.93 (s, 1H), 6.85 (s, 1H), 6.81 (d, 1H, J = 8 Hz), 6.77 (d, 1H, J = 11 Hz), 6.49 (s, 1H), 6.45 (dd, 1H, J = 15 Hz, 11 Hz), 6.34 (d, 1H, J = 7 Hz), 6.28 (s, 1H), 5.74 (dd, 1H, J = 15 Hz, 9 Hz), 5.38 (m, 1H), 4.90 (m, 1H), 4.32 (t, 1H, J = 11 Hz), 4.00 (s, 3H), 3.69 (d, 1H, J = 13 Hz), 3.51 (d, 1H, J = 9 Hz), 3.36 (s, 3H), 3.15 (m, 1H), 3.12 (s, 3H), 3.01 (d, 1H, J = 10 Hz), 2.73 (s, 3H), 2.69 (m, 1H), 2.22 (m, 1H), 2.18 (s, 3H), 1.68 (m, 1H), 1.66 (s, 3H), 1.51 (m, 1H), 1.47 (d, 3H, J = 7 Hz), 1.30 (d, 3H, J = 6 Hz), 1.28 (m, 1H), 0.87 (s, 3H). Example 18
[0505] Compound 53 was synthesized as described below and as depicted in FIG 23.
Maytansin-N-methyl-L-alanine-(3-methyl-4-amino)benzamide (53) Step A: Maytansin-N-methyl-L-alanine-(3-methyl-4-nitro)benzamide :
[0506] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 49 mg, 0.075 mmol) and 3-methyl-4-nitrobenzoic acid (52) (26 mg, 0.143 mmol), using the method from Step A of Example 5, to give a yellow solid (34 mg, 56%). MS (ESI, pos.): calc’d for C40H49N4O12Cl, 812.3; found 795.2 (M-H2O+H), 813.2 (M+H). Step B: Maytansin-N-methyl-L-alanine-(3-methyl-4-amino)benzamide (53):
[0507] The title compound was prepared from the product of the preceding step (33 mg, 0.041 mmol), using the method from Step B of Example 5, to give a white solid (24 mg, 71%). MS (ESI, pos.): calc’d for C40H51N4O10Cl, 782.3; found 765.2 (M-H2O+H), 783.2 (M+H). 1H-NMR (500 MHz, CDCl3): d 7.15 (s, 1H), 7.09 (d, 1H, J = 8 Hz), 6.94 (s, 1H), Hz), 6.33 (s, 1H), 5.75 (dd, 1H, J = 14 Hz, 9 Hz), 5.42 (m, 1H), 4.87 (dd, 1H, J = 12 Hz, 10 Hz), 4.31 (t, 1H, J = 11 Hz), 3.98 (s, 3H), 3.63 (br d, 1H, J = 13 Hz), 3.50 (d, 1H, J = 9 Hz), 3.36 (s, 3H), 3.10 (m, 1H), 3.07 (s, 3H), 3.03 (d, 1H, J = 10 Hz), 2.91 (s, 3H), 2.68 (t, 1H, J = 13 Hz), 2.19 (dd, 1H, J = 14 Hz, 3 Hz), 2.06 (s, 3H), 1.66 (m, 1H), 1.65 (s, 3H), 1.48 (dd, 1H, J = 11 Hz, 7 Hz), 1.43 (d, 3H, J = 7 Hz), 1.29 (d, 3H, J = 7 Hz), 1.27 (m, 1H), 0.84 (s, 3H). Example 19
[0508] Compound 55 was synthesized as described below and as depicted in FIG 24.
Maytansin-N-methyl-L-alanine-(8-amino-5-carboxyquinoline)carboxamide (55) Step A: Maytansin-N-methyl-L-alanine-(8-nitro-5-carboxyquinoline)carboxamide :
[0509] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 47 mg, 0.072 mmol) and 8-nitro-5-carboxyquinoline (54) (35 mg, 0.160 mmol), using the method from Step A of Example 5, to give a pale yellow solid (30 mg, 49%). MS (ESI, pos.): calc’d for C42H48N5O12Cl, 849.3; found 832.6 (M-H2O+H), 850.7 (M+H). Step B: Maytansin-N-methyl-L-alanine-(8-amino-5-carboxyquinoline)carboxamide (55):
[0510] The title compound was prepared from the product of the preceding step (29 mg, 0.034 mmol), using the method from Step B of Example 5, to give a pale yellow solid (18 mg, 60%). MS (ESI, pos.): calc’d for C42H50N5O10Cl, 819.3; found 802.0 (M-H2O+H), 820.0 (M+H). 1H-NMR (500 MHz, CDCl3): d 8.77 (dd, 1H, J = 4 Hz, 2 Hz), 8.18 (d, 1H, J = 8 Hz), 7.40 (dd, 1H, J = 9 Hz, 4 Hz), 7.23 (m, 1H), 6.99 (s, 1H), 6.87 (s, 1H), 6.73 (d, 1H, J = 8 Hz), 6.68 (d, 1H, J = 11 Hz), 6.47 (dd, 1H, J = 15 Hz, 11 Hz), 6.26 (s, 1H), 5.77 (dd, 1H, J = 15 Hz, 9 Hz), 5.32 (br m, 1H), 5.21 (br s, 2H), 4.99 (d, 1H, J = 9 Hz), 4.34 (t, 1H, J = 11 Hz), 4.01 (s, 3H), 3.78 (br m, 1H), 3.67 (d, 1H, J = 13 Hz), 3.51 (d, 1H, J = 10 Hz), 3.37 (s, 3H), 3.16 (d, 1H, J = 13 Hz), 3.12 (s, 3H), 3.00 (d, 1H, J = 10 Hz), 2.79 (s, 3H), 2.73 (m, 1H), 2.24 (dd, 1H, J = 14 Hz, 3 Hz), 1.76 (d, 1H, J = 14 Hz), 1.68 (s, 3H), 1.61 (d, 3H, J = 7 Hz), 1.33 (m, 1H), 1.30 (d, 3H, J = 7 Hz), 1.25 (s, 1H), 0.90 (s, 3H). Example 20
[0511] Compound 60 was synthesized as described below and as depicted in FIG 25. Maytansin-N-methyl-L-alanine‐(3‐methoxy‐4‐amino)benzamido‐Cit‐Val‐Cap‐Mal^(60) Step A: Boc‐L‐valine‐L‐citrulline‐(3‐methoxy‐4‐amino)benzoic^acid^t‐butyl^ester^(57):
[0512] The title compound was prepared from Boc-L-valine-L-citrulline (3, 100 mg, 0.267 mmol) and 4-amino-3-methoxybenzoic acid tert-butyl ester (56, 61 mg, 0.273 mmol), using the method from Step A of Example 2, to give a white solid (74 mg, 48%). MS (ESI, pos.): calc’d for C28H45N5O8, 579.3; found 580.4 (M+H), 602.6 (M+Na). Step B: L‐valine‐L‐citrulline‐(3‐methoxy‐4‐amino)benzoic^acid^(58):
[0513] The title compound was prepared from the product of the preceding step (57, 72 mg, 0.124 mmol), using the method from Step C of Example 1, to give an off-white solid (68 mg, 100%). MS (ESI, pos.): calc’d for C19H29N5O6, 423.2; found 424.4 (M+H), 847.4 (2M+H). Step C: 6-(Maleimidyl-caprolyl)-L‐valine‐L‐citrulline‐(3‐methoxy‐4‐amino)benzoic^ acid^ (59):
[0514] The title compound was prepared from the product of the preceding step (58, 67 mg, 0.124 mmol), using the method from Step D of Example 1, to give a white solid (45 mg, 59%). MS (ESI, pos.): calc’d for C29H40N6O9, 616.3; found 617.5 (M+H), 639.6 (M+Na). Step D: Maytansin-N-methyl-L-alanine‐(3‐methoxy‐4‐amino)benzamido‐Cit‐Val‐Cap‐Mal^ (60):
[0515] The title compound was prepared from the product of the preceding step (59, 44 mg, 0.071 mmol) and maytansin-N-methyl-L-alanine (9, 49 mg, 0.075 mmol), using the method from Step E of Example 1, to give a white solid (14 mg, 16%). MS (ESI, pos.): calc’d for C61H82N9O17Cl, 1247.6; found 1231.1 (M-H2O+H), 1249.1 (M+H), 1271.1 (M+Na). 1H-NMR (500 MHz, CDCl3): d 8.48 (s, 1H), 8.24 (d, 1H, J = 8 Hz), 7.11 (d, 1H, J = 8 Hz), 6.96– 6.93 (m, 3H), 6.83 (s, 1H), 6.72– 6.68 (m, 3H), 6.45 (dd, 1H, J = 16 Hz, 11 Hz), 6.25 (s, 1H), 6.18 (d, 1H, J = 9 Hz), 5.77 (dd, 1H, J = 15 Hz, 10 Hz), 5.44 (m, 1H), 5.03 (br s, 1H), 4.90 (d, 1H, J = 10 Hz), 4.62 (m, 1H), 4.54 (br s, 2H), 4.33– 4.28 (m, 2H), 3.99 (s, 3H), 3.75 (s, 3H), 3.61 (d, 1H, J = 13 Hz), 3.52– 3.48 (m, 3H), 3.36 (s, 3H), 3.24 (m, 2H), 3.11 (d, 1H, J = 13 Hz), 3.07 (s, 3H), 3.03 (d, 1H, J = 10 Hz), 2.90 (s, 3H), 2.70 (m, 1H), 2.26 = 7 Hz), 1.33– 1.25 (m, 6H), 0.95 (m, 6H), 0.86 (s, 3H).
Example 21
[0516] Compound 63 was synthesized as described below and as depicted in FIG 26.
Maytansin-N-methyl-L-alanine‐(2‐fluoro‐4‐amino)benzamido‐Cit‐Val‐Cap‐6‐amine^(63) Step A: Boc-6-aminohexanoic acid succinate ester:
[0517] The title compound was prepared from Boc-6-aminohexanoic acid (64, 502 mg, 2.17 mmol), using the method from Step A of Example 1, to give a white solid (712 mg, 99%). MS (ESI, pos.): calc’d for C15H24N2O6, 328.2; found 351.2 (M+Na). Step B: Boc-(6-amino-caprolyl)-L‐valine‐L‐citrulline (62):
[0518] The title compound was prepared from the product of the preceding steps (710 mg, 2.16 mmol) and L-valine-L-citrulline TFA salt (970 mg, 2.51 mmol), using the method from Step D of Example 1, to give a pale gold solid (720 mg, 69%). MS (ESI, pos.): calc’d for C22H41N5O7, 487.3; found 488.3 (M+H), (M+H). Step C: Maytansin-N-methyl-L-alanine‐(2‐fluoro‐4‐amino)benzamido‐Cit‐Val‐Cap‐6‐Boc‐ amine:
[0519] The title compound was prepared from the product of the preceding step (62, 25 mg, 0.051 mmol) and Maytansin-N-methyl-L-alanine-(2-fluoro-4-amino)benzamide (29, 35 mg, 0.041 mmol), using the method from Step A of Example 2, to give a white solid (17 mg, 33%). MS (ESI, pos.): calc’d for C61H87N9O16ClF, 1255.6; found 1238.5 (M-H2O+H), 1256.6 (M+H), 1278.6 (M+Na). Step D: Maytansin-N-methyl-L-alanine-(2-fluoro-4-amino)benzamido-Cit-Val-Cap-6-amine^ (63):
[0520] The product of the preceding step (16 mg, 0.013 mmol) was dissolved in acetonitrile (MeCN, 3 mL) and water (1 mL), treated with trifluoroacetic acid (TFA, 1.0 mL, 13.0 mmol), the flask sealed with a rubber septum, purged with argon, and the reaction stirred at ambient temperature. After 24 h, the reaction was partially concentrated in vacuo at ambient temperature, diluted with water (ca. 1 mL), and purified twice on C18 Aq RediSep purest fractions by LCMS were combined, partially concentrated in vacuo at ambient temperature, frozen at -78oC, and lyophilized to give the title compound as a white solid (9 mg, 56%). MS (ESI, pos.): calc’d for C56H79N9O14ClF, 1155.5; found 1156.6 (M+H), 1178.6 (M+Na). 1H-NMR (500 MHz, CD3OD): d 8.72 (d, 1H, J = 12 Hz), 8.39 (d, 1H, J = 13 Hz), 8.14 (d, 1H, J = 10 Hz), 7.84 (dd, 1H, J = 21 Hz, 3 Hz), 7.75 (dd, 1H, J = 21 Hz, 3 Hz), 7.60 (dd, 1H, J = 15 Hz, 3 Hz), 7.33 (dd, 1H, J = 14 Hz, 3 Hz), 7.25 (m, 1H), 7.21 (s, 1H), 6.97 (s, 1H), 6.75 (m, 1H), 6.72 (s, 1H), 6.67 (m, 1H), 5.78– 5.60 (m, 3H), 4.77 (m, 1H), 4.45 (m, 2H), 4.26 (m, 1H), 4.20 (d, 1H, J = 13 Hz), 4.05– 4.00 (m, 2H), 4.03 (s, 3H), 3.69 (dd, 1H, J = 21 Hz, 5 Hz), 3.64 (d, 1H, J = 16 Hz), 3.41 (s, 3H), 3.30– 3.26 (m, 2H), 3.23– 3.09 (m, 2H), 3.05 (m, 3H), 2.97 (dd, 1H, J = 16 Hz, 5 Hz), 2.92 (d, 1H, J = 13 Hz), 2.86 (m, 3H), 2.82 (d, 1H, J = 13 Hz), 2.75 (m, 1H), 2.36– 2.29 (m, 2H), 2.25– 2.20 (m, 1H), 2.08– 2.02 (m, 2H), 1.76 (s, 3H), 1.73– 1.50 (m, 10H), 1.45 (m, 2H), 1.36– 1.32 (m, 2H), 1.28 (d, 3H, J = 11 Hz), 1.05 (d, 3H, J = 11 Hz), 1.03– 0.98 (m, 4H), 0.93 (s, 3H).
[0521] Example 22
[0522] Compound 65 was synthesized as described below and as depicted in FIG 27. Maytansin-N-methyl-L-alanine-N-(2-methoxy-5-amino)benzamide (65) Step A: Maytansin-N-methyl-L-alanine-(2-methoxy-5-nitro)benzamide:
[0523] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 50 mg 0.077 mmol) and 2-methoxy-5-nitrobenzoic acid (64) (25 mg, 0.127 mmol), using the method from Step A of Example 5, to give a white solid (51 mg, 80%). MS (ESI, pos.): calc’d for C40H49ClN4O13, 829.3; found 812.0 (M-H2O+H), 830.0 (M+H), 852.0 (M+Na). Step B: Maytansin-N-methyl-L-alanine-N-(2-methoxy-5-amino)benzamide (65):
[0524] The title compound was prepared from the product of the preceding step (50 mg, 0.060 mmol), using the method from Step B of Example 5, to give a white solid (19 mg, 40%). MS (ESI, pos.): calc’d for C40H51ClN4O11, 798.3; found 781.3 (M-H2O), 799.3 (M+H), 822.3 (M+Na). Example 23
[0525] Compound 67 was synthesized as described below and as depicted in FIG 28. Maytansin-N-methyl-L-alanine-N-(3-amino-4-methoxy)benzamide (67) Step A: Maytansin-N-methyl-L-alanine-(3-nitro-4-methoxy)benzamide:
[0526] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 50 mg 0.077 mmol) and 3-nitro-4-methoxybenzoic acid (66) (25 mg, 0.127 mmol), using the method from Step A of Example 5, to give a white solid (46 mg, 72%). MS (ESI, pos.): calc’d for C40H49ClN4O13, 829.3; found 812.0 (M-H2O+H), 830.0 (M+H), 852.0 (M+Na). Step B: Maytansin-N-methyl-L-alanine-N-(3-amino-4-methoxy)benzamide (67):
[0527] The title compound was prepared from the product of the preceding step (45 mg, 0.054 mmol), using the method from Step B of Example 5, to give a white solid (23 mg, 53%). MS (ESI, pos.): calc’d for C40H51ClN4O11, 798.3; found 781.3 (M-H2O), 799.3 (M+H), 822.3 (M+Na). Example 24
[0528] Compound 69 was synthesized as described below and as depicted in FIG 29. Maytansin-N-methyl-L-alanine-N-(3-amino-5-fluoro)benzamide (69)
Step A: Maytansin-N-methyl-L-alanine-(3-nitro-5-fluoro)benzamide:
[0529] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 50 mg, 0.077 mmol) and 3-nitro-5-fluorobenzoic acid (68) (24 mg, 0.127 mmol), using the method from Step A of Example 5, to give a yellow solid (37 mg, 59%). MS (ESI, pos.): calc’d for C39H46ClFN4O12, 816.3; found 817.2 (M+H).
Step B: Maytansin-N-methyl-L-alanine-N-(3-amino-5-fluoro)benzamide (69):
[0530] The title compound was prepared from the product of the preceding step (37 mg, 0.045 mmol), using the method from Step B of Example 5, to give a white solid (9.1 mg, 24%). MS (ESI, pos.): calc’d for C39H48ClFN4O10, 786.3; found 769.3 (M-H2O), 787.3 (M+H). [0531] Compound 71 was synthesized as described below and as depicted in FIG 30.
Maytansin-N-methyl-L-alanine-N-(2-fluoro-5-amino)benzamide (71) Step A: Maytansin-N-methyl-L-alanine-(2-fluoro-5-nitro)benzamide:
[0532] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 50 mg, 0.077 mmol) and 2-flouro-5-nitrobenzoic acid (70) (24 mg, 0.127 mmol), using the method from Step A of Example 5, to give a yellow solid (37 mg, 59%). MS (ESI, pos.): calc’d for C39H46ClFN4O12, 816.3; found 799.3 (M-H2O), 817.2 (M+H). Step B: Maytansin-N-methyl-L-alanine-N-(2-fluoro-5-amino)benzamide (71):
[0533] The title compound was prepared from the product of the preceding step (37 mg, 0.045 mmol), using the method from Step B of Example 5, to give a white solid (2.2 mg, 6%). MS (ESI, pos.): calc’d for C39H48ClFN4O10, 786.3; found 769.3 (M-H2O), 787.3 (M+H). Example 26
[0534] Compound 73 was synthesized as described below and as depicted in FIG 31.
Maytansin-N-methyl-L-alanine-N-(3-amino)benzamide (73) Step A: Maytansin-N-methyl-L-alanine-(3-nitro)benzamide:
[0535] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 50 mg, 0.077 mmol) and 3-nitrobenzoic acid (72) (21 mg, 0.127 mmol), using the method from Step A of Example 5, to give a white solid (34 mg, 56%). MS (ESI, pos.): calc’d for C39H47ClN4O12, 798.3; found 781.2 (M-H2O), 799.3 (M+H). [0536] Step B: Maytansin-N-methyl-L-alanine-N-(3-amino)benzamide (73):
[0537] The title compound was prepared from the product of the preceding step (34 mg, 0.043 mmol), using the method from Step B of Example 5, to give a white solid (9.3 mg, 27%). MS (ESI, pos.): calc’d for C39H49ClN4O10, 768.3; found 751.2 (M-H2O), 769.2 (M+H). [0538] Compound 75 was synthesized as described below and as depicted in FIG 32. Maytansin-N-methyl-L-alanine-N-(3-amino-4-fluoro)benzamide (75) Step A: Maytansin-N-methyl-L-alanine-(3-nitro-4-fluoro)benzamide:
[0539] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 50 mg, 0.077 mmol) and 3-nitro-4-fluorobenzoic acid (74) (24 mg, 0.127 mmol), using the method from Step A of Example 5, to give a white solid (34 mg, 54%). MS (ESI, pos.): calc’d for C39H46ClFN4O12, 816.3; found 799.3 (M-H2O), 817.2 (M+H). [0540] Step B: Maytansin-N-methyl-L-alanine-N-(3-amino-4-fluoro)benzamide (75):
[0541] The title compound was prepared from the product of the preceding step (34 mg, 0.042 mmol), using the method from Step B of Example 5, to give a white solid (12 mg, 37%). MS (ESI, pos.): calc’d for C39H48ClFN4O10, 786.3; found 769.3 (M-H2O), 787.3 (M+H). Example 28
[0542] Compound 77 was synthesized as described below and as depicted in FIG 33.
Maytansin-N-methyl-L-alanine-N-4-aminobenzamide-adipic-NHS (77) Step A: Maytansin-N-methyl-L-alanine-N-(4-amino)benzamide-adipic acid (76):
[0543] The title compound was prepared from 27 (20 mg, 0.026 mmol) and adipic anhydride (17 mg, 0.133 mmol) were weighed into a round-bottom flask and dissolved in pyridine (1.5 mL). The flask was sealed via rubber septum, purged with nitrogen, and the reaction was stirred at ambient temperature. After 4 h the reaction was purified directly on a 30 g C18 RediSep Gold Aq column via ISCO system (gradient elution: 20– 90% MeCN in water, 0.05% acetic acid in both, over 20 min). The product-containing fractions were combined, partially concentrated invacuo, frozen on dry ice, and lyophilized to give an off- white solid (16 mg, 67%). MS (ESI, pos.): calc’d for C45H57ClN4O13, 896.4; found 879.4 (M- H2O), 897.4 (M+H). [0545] The title compound was prepared from the product of the preceding step (76, 16 mg, 0.017 mmol), using the method from Step A of Example 1, to give a white solid (10 mg, 58%). MS (ESI, pos.): calc’d for C49H60ClN5O15, 993.5; found 976.0 (M-H2O), 994.0 (M+H). 1H-NMR (500 MHz, CDCl3): δ 7.92 (s, 1H), 7.57 (d, 2H, J = 8 Hz), 7.36 (d, 2H, J = 8 Hz), 6.93 (s, 1H), 6.86 (s, 1H), 6.75 (d, 1H, J = 12 Hz), 6.47 (dd, 1H, J = 15 Hz, 11 Hz), 6.29 (s, 1H), 5.74 (dd, 1H, J = 16 Hz, 9 Hz), 5.47 (m, 1H), 4.90 (dd, 1H, J = 12 Hz, 3 Hz), 4.32 (t, 1H, J = 11 Hz), 4.02 (s, 4H), 3.63 (d, 1H, J = 13 Hz), 3.62 (br s, 1H), 3.53 (d, 1H, J = 9 Hz), 3.38 (s, 3H), 3.13 (d, 1H, J = 13 Hz), 2 (s, 3H), 2.80 (d, 2H, J = 10 Hz), 2.73 (s, 3H), 2.60 (m, 1H), 2.27 (t, 2H, J = 10 Hz), 2.11 (br s, 1H), 2.07 (s, 2H), 1.62 (s, 3H), 1.58– 1.52 (m, 2H), 1.51– 1.46 (m, 2H), 1.34– 1.29 (m, 3H), 1.25– 1.20 (m, 2H), 1.13 (d, 3H, J = 7 Hz), 0.82 (s, 3H). Example 29
[0546] Compound 78 was synthesized as described below and as depicted in FIG 34.
Maytansin-N-methyl-L-alanine-4-aminobenzamide-Cap-Mal (78)
[0547] The title compound was prepared from 27 (15 mg, 0.019 mmol) and 6- maleimidohexanoic acid (6 mg, 0.029 mmol), using the method from Step A of Example 5, to give an off-white solid (9.8 mg, 52%). MS (ESI, pos.): calc’d for C49H60ClN5O13, 962.5; found 944.8 (M-H2O), 962.8 (M+H). 1H-NMR (500 MHz, DMSO-d6): δ 10.01 (s, 1H), 7.56 (d, 2H, J = 9 Hz), 7.29 (d, 2H, J = 9 Hz), 7.21 (s, 1H), 6.99 (s, 2H), 6.91 (s, 1H), 6.83 (s, 1H), 6.62-6.56 (m, 2H), 5.97 (s, 1H), 5.61 (dd, 1H, J = 15 Hz, 10 Hz), 5.44 (m, 1H), 4.59 (d, 1H, J = 12 Hz), 4.10 (t, 1H, J = 12 Hz), 3.94 (s, 3H), 3.51 (d, 1H, J = 9 Hz), 3.40 - 3.36 (m, 2H), 3.26 (s, 3H), 3.23– 3.21 (m, 2H), 2.94 (s, 3H), 2.80 (d, 1H, J = 10 Hz), 2.73 (s, 3H), 2.64– 2.57 (m, 1H), 2.27 (t, 2H, J = 10 Hz), 2.11– 2.09 (m, 1H), 2.07 (s, 2H), 1.62 (s, 3H), 1.57– 1.52 (m, 2H), 1.51– 1.46 (m, 2H), 1.34– 1.29 (m, 3H), 1.25– 1.19 (m, 2H), 1.13 (d, 3H, J = 7 Hz), 0.82 (s, 3H).
Example 30
[0548] Compound 80 was synthesized as described below and as depicted in FIG 35. Step A: Maytansin-N-methyl-L-alanine-(3-methylsulfonyl-4-nitro)benzamide:
[0549] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 40 mg, 0.062 mmol) and 3-methylsulfonyl-4-nitrobenzoic acid (79) (25 mg, 0.102 mmol), using the method from Step A of Example 5, to give a yellow solid (37 mg, 69%). MS (ESI, pos.): calc’d for C50H49N4O14ClS, 876.3; found 857.6 (M-H2O+H), 875.6 (M+H), 897.6 (M+Na). Step B: Maytansin-N-methyl-L-alanine-(3-methylsulfonyl-4-amino)benzamide (80):
[0550] The title compound was prepared from the product of the preceding step (36 mg, 0.041 mmol), using the method from Step B of Example 5, to give a white solid (28 mg, 76%). MS (ESI, pos.): calc’d for C40H51N4O12ClS, 846.3; found 829.1 (M-H2O+H), 847.1 (M+H), 869.1 (M+Na). 1H-NMR (500 MHz, CDCl3): ^ 7.85 (s, 1H), 7.44 (d, 1H, J = 9 Hz), 6.90 (s, 1H), 6.84 (s, 1H), 6.71 (d, 1H, J = 9 Hz), 6.66 (d, 1H, J = 11 Hz), 6.45 (dd, 1H, J = 15 Hz, 11 Hz), 6.23 (s, 1H), 5.72 (dd, 1H, J = 15 Hz, 10 Hz), 5.32– 5.25 (m, 2H), 4.89 (dd, 1H, J = 12 Hz, 3 Hz), 4.31 (t, 1H, J = 11 Hz), 3.99 (s, 3H), 3.65 (d, 1H, J = 13 Hz), 3.51 (d, 1H, J = 9 Hz), 3.36 (s, 3H), 3.14 (d, 1H, J = 13 Hz), 3.05 (s, 3H), 3.01 (m, 1H), 2.99 (s, 3H), 2.95 (s, 3H), 2.68 (t, 1H, J = 14 Hz), 2.20 (dd, 1H, J = 15 Hz, 3 Hz), 1.70 (m, 1H), 1.67 (s, 3H), 1.55 (s, 3H), 1.52 (m, 1H), 1.47 (d, 1H, J = 7 Hz), 1.31 (d, 1H, J = 7 Hz), 1.28 (m, 1H), 0.85 (s, 3H).
Example 31
[0551] Compound 82 was synthesized as described below and as depicted in FIG 36.
Maytansin-N-methyl-L-alanine-N-(3-hydroxy-4-amino)benzamide (82) Step A: Maytansin-N-methyl-L-alanine-(3-hydroxy-4-nitro)benzamide:
[0552] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 47 mg, 0.072 mmol) and 3-hydroxy-4-nitrobenzoic acid (81) (20 mg, 0.109 mmol), using the method from Step A of Example 5, to give a yellow solid (29 mg, 49%). MS (ESI, pos.): calc’d for C39H47N4O13Cl, 814.3; found 796.8 (M-H2O+H), 814.8 (M+H), 836.8 (M+Na). [0553] The title compound was prepared from the product of the preceding step (28 mg, 0.034 mmol), using the method from Step B of Example 5, to give a pale yellow solid (20 mg, 69%). MS (ESI, pos.): calc’d for C40H51N4O12ClS, 784.3; found 767.7 (M-H2O+H), 785.7 (M+H). 1H-NMR (500 MHz, CDCl3): ^ ^9.19 (s, 1H), 7.17 (s, 1H), 6.88 (s, 1H), 6.79 (br s, 2H), 6.63– 6.56 (m, 3H), 6.47 (m, 1H), 5.93 (s, 1H), 4.93 (s, 2H), 4.57 (d, 1H, J = 11 Hz), 4.09 (t, 1H, J = 12 Hz), 3.93 (s, 3H), 3.50 (d, 1H, J = 9 Hz), 3.26 (s, 3H), 3.18 (br m, 1H), 2.99 (s, 3H), 2.79 (d, 1H, J = 10 Hz), 2.76 (s, 3H), 2.06 (dd, 1H, J = 14 Hz, 2 Hz), 1.60 (s, 3H), 1.48– 1.46 (m, 2H), 1.33– 1.27 (m, 4H), 1.13 (d, 3H, J = 6 Hz), 0.80 (s, 3H).
Example 32
[0554] Compound 84 was synthesized as described below and as depicted in FIG 37.
Maytansin-N-methyl-L-alanine-N-(2-amino)benzamide (84) Step A: Maytansin-N-methyl-L-alanine-(2-nitro)benzamide:
[0555] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 30 mg, 0.046 mmol) and 2-nitrobenzoic acid (83) (15 mg, 0.092 mmol), using the method from Step A of Example 5, to give a yellow solid (26 mg, 71%). MS (ESI, pos.): calc’d for C39H47N4O12Cl, 798.3; found 799.3 (M+H), 821.3 (M+Na).
Step B: Maytansin-N-methyl-L-alanine-(2-amino)benzamide (84):
[0556] The title compound was prepared from the product of the preceding step (24 mg, 0.038 mmol), using the method from Step B of Example 5, to give a white solid (13 mg, 54%). MS (ESI, pos.): calc’d for C39H49N4O10Cl, 768.3; found 769.0 (M+H). 1H-NMR (500 MHz, CDCl3): ^ ^7.16 (m, 1H), 7.08 (d, 1H, J = 6 Hz), 6.92 (s, 1H), 6.84 (d, 1H, J = 2 Hz), 6.71 (d, 1H, J = 8 Hz), 6.63 (t, 1H, J = 7 Hz), 6.57 (m, 1H), 6.47 (dd, 1H, J = 15 Hz, 11 Hz), 6.27 (s, 1H), 5.76 (m, 1H), 5.20 (br s, 1H), 4.99 (m, 1H), 4.39– 4.32 (m, 3H), 4.01 (s, 3H), 3.74 (br s, 1H), 3.59 (d, 1H, J = 13 Hz), 3.52 (d, 1H, J = 9 Hz), 3.39 (s, 3H), 3.20 (s, 3H), 3.15 (d, 1H, J = 12 Hz), 2.99 (d, 1H, J = 12 Hz), 2.94 (s, 3H), 2.73 (t, 1H, J = 14 Hz), 2.26 (m, 1H), 1.75 (d, 1H, J = 12 Hz), 1.69 (s, 3H), 1.55 (d, 3H, J = 7 Hz), 1.50 (m, 1H), 1.35– 1.30 (m, 4H), 0.90 (s, 3H). Example 33
[0557] Compound 86 was synthesized as described below and as depicted in FIG 38.
Maytansin-N-methyl-L-alanine-N-(4-methoxy-2-amino)benzamide (86) Step A: Maytansin-N-methyl-L-alanine-(4-methoxy-2-nitro)benzamide:
[0558] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 30 mg, 0.046 mmol) and 4-methoxy-2-nitrobenzoic acid (85) (18 mg, 0.092 mmol), using the method from Step A of Example 5, to give a yellow solid (18 mg, 47%). MS (ESI, pos.): calc’d for C40H49N4O13Cl, 828.3; found 829.4 (M+H), 851.3 (M+Na).
Step B: Maytansin-N-methyl-L-alanine-(4-methoxy-2-amino)benzamide (86):
[0559] The title compound was prepared from the product of the preceding step (18 mg, 0.022 mmol), using the method from Step B of Example 5, to give a white solid (15 mg, 84%). MS (ESI, pos.): calc’d for C40H51N4O11Cl, 798.3; found 799.1 (M+H). 1H-NMR (500 MHz, CDCl3): ^ ^6.94 (s, 1H), 6.85 (d, 1H, J = 2 Hz), 6.79 (dd, 1H, J = 9 Hz, 3 Hz), 6.67 (d, 1H, J = 9 Hz), 6.65 (m, 1H), 6.60 (d, 1H, J = 11 Hz), 6.48 (dd, 1H, J = 15 Hz, 12 Hz), 6.27 (s, 1H), 5.76 (m, 1H), 5.22 (br s, 1H), 4.98 (m, 1H), 4.36 (t, 1H, J = 11 Hz), 4.01 (s, 3H), 3.93 (br s, 1H), 3.72 (br s, 1H), 3.65 (m, 1H), 3.61 (s, 3H), 3.52 (d, 1H, J = 9 Hz), 3.39 (s, 3H), 3.18 (s, 3H), 3.16 (m, 1H), 2.99 (d, 1H, J = 10 Hz), 2.94 (s, 3H), 2.72 (t, 1H, J = 13 Hz), 2.26 (dd, 1H, J = 15 Hz, 3 Hz), 1.74 (d, 1H, J = 14 Hz), 1.70 (s, 3H), 1.55 (d, 3H, J = 7 Hz), 1.52 – 1.48 (m, 1H), 1.35– 1.30 (m, 4H), 0.90 (s, 3H).
Example 34
[0560] Compound 88 was synthesized as described below and as depicted in FIG 39.
Maytansin-N-methyl-L-alanine-N-(3-morpholino-4-amino)benzamide (88) Step A: Maytansin-N-methyl-L-alanine-(3-morpholino-4-nitro)benzamide: mg, 0.046 mmol) and 3-morpholino-4-nitrobenzoic acid (87) (23 mg, 0.092 mmol), using the method from Step A of Example 5, to give a yellow solid (28 mg, 70%). MS (ESI, pos.): calc’d for C43H54N5O13Cl, 883.3; found 884.5 (M+H), 906.3 (M+Na).
Step B: Maytansin-N-methyl-L-alanine-(3-morpholino-4-amino)benzamide (88):
[0562] The title compound was prepared from the product of the preceding step (28 mg, 0.032 mmol), using the method from Step B of Example 5, to give an off-white solid (12 mg, 52%). MS (ESI, pos.): calc’d for C43H56N5O11Cl, 853.4; found 853.9 (M+H), 875.9 (M+Na). 1H-NMR (500 MHz, CDCl3): ^ ^7.12 (s, 1H), 7.02 (d, 1H, J = 8 Hz), 6.98 (s, 1H), 6.86 (d, 1H, J = 2 Hz), 6.81 (d, 1H, J = 11 Hz), 6.61 (d, 1H, J = 8 Hz), 6.48 (dd, 1H, J = 16 Hz, 12 Hz), 6.29 (s, 1H), 5.77 (dd, 1H, J = 15 Hz, 9 Hz), 5.48 (m, 1H), 4.89 (dd, 1H, J = 12 Hz, 3 Hz), 4.34 (t, 1H, J = 12 Hz), 4.19 (br m, 1H), 4.01 (s, 3H), 3.80– 3.75 (m, 5H), 3.53 (m, 2H), 3.39 (s, 3H), 3.15 (d, 1H, J = 13 Hz), 3.07 (d, 1H, J = 10 Hz), 3.03 (s, 3H), 2.96 (s, 3H), 2.73 (m, 5H), 2.22 (dd, 1H, J = 14 Hz, 3 Hz), 1.71 (m, 1H), 1.69 (s, 3H), 1.54– 1.49 (m, 1H), 1.47 (d, 3H, J = 8 Hz), 1.33 (d, 3H, J = 7 Hz), 1.30 (m, 1H), 0.88 (s, 3H).
Example 35
[0563] Compound 90 was synthesized as described below and as depicted in FIG 40.
Maytansin-N-methyl-L-alanine-N-(3-acetamido-4-amino)benzamide (90) Step A: Maytansin-N-methyl-L-alanine-(3-acetamido-4-nitro)benzamide:
[0564] The title compound was prepared from maytansin-N-methyl-L-alanine (9, 50 mg, 0.077 mmol) and 3-acetamido-4-nitrobenzoic acid (89) (29 mg, 0.129 mmol), using the method from Step A of Example 5, to give a fluffy pale yellow solid (36 mg, 54%). MS (ESI, pos.): calc’d for C41H50N5O13Cl, 855.3; found 839.1 (M-H2O), 857.1 (M+H), 879.1 (M+Na).
Step B: Maytansin-N-methyl-L-alanine-(3-acetamido-4-amino)benzamide (90): mg, 0.042 mmol), using the method from Step B of Example 5, to give a white solid (19 mg, 57%). MS (ESI, pos.): calc’d for C41H52N5O11Cl, 825.3; found 808.4 (M-H2O), 826.4 (M+H). 1H-NMR (500 MHz, DMSO-d6): ^ ^9.05 (s, 1H), 7.30 (s, 1H), 7.17 (s, 1H), 6.98 (d, 1H, J = 9 Hz), 6.91 (s, 1H), 6.79 (s, 1H), 6.58 (m, 3H), 5.97 (s, 1H), 5.64 (br s, 1H), 5.34 (m, 3H), 4.60 (d, 1H, J = 12 Hz), 4.10 (t, 1H, J = 12 Hz), 3.93 (s, 3H), 3.50 (d, 1H, J = 9 Hz), 3.33 (s, 3H), 3.29 (m, 1H), 3.26 (s, 3H), 3.16 (d, 1H, J = 12 Hz), 2.97 (s, 3H), 2.79 (m, 4H), 2.07 (d, 1H, J = 13 Hz), 1.95 (s, 2H), 1.59 (s, 3H), 1.51– 1.42 (m, 2H), 1.29 (m, 3H), 1.13 (d, 3H, J = 6 Hz), 0.81 (s, 3H).
Example 36
[0566] Compound 100 was synthesized as described below and as depicted in FIG 41.
Maytansin-N-methyl-L-alanine-N-4-aminobenzamide-Cit-Val-cap-diBromomethylacryl (100) Step A: 3-Bromo-2-bromomethyl-propionyl chloride (92):
[0567] To a 10 mL round bottom flask equipped with a magnetic stirrer and nitrogen inlet was charged 3-romo-2-bromomethyl-propionic acid (91, 1.0 g; 4.1 mmol) and thionyl chloride (3.0 mL). This solution was heated to reflux for 3 hours and concentrated to 0.90 g (84% yield) as a brown oil. 1H-NMR (300 MHz, CDCl3): ^ 3.85– 3.75 (m, 4H), 3.60 (pentet, 1H, J = 9 Hz).
Step B: 6-Amino-hexanoic acid tert-butyl ester (94):
[0568] To a 50 mL round bottom flask equipped with a magnetic stirrer and nitrogen inlet was charged 6-aminohexanoic acid (93, 2.0 g; 15 mmol) and thionyl chloride (5.0 mL; 69 mmol; 4.5 equiv.). This solution was stirred at or below 30 oC for 2 hours and concentrated in vacuo to dryness. To the tan semi-solid a slurry of sodium bicarbonate (2.6 g; 30 mmol; 2.0 equiv.) in t-BuOH (5.0 mL; 87 mmol; 5.7 equiv.) was added and the slurry was stirred at ambient temperature for another 2 h. The butanol was removed in vacuo at 40 oC. The thick white slurry was diluted with ethyl acetate and washed with 4 portions of 1 N NaOH, 3 portions of H2O, 1 portion of brine. The organics were dried over Na2SO4, filtered C10H21NO2, 187.3; found 188.4 (M+H), 1H-NMR (300 MHz, CDCl3): ^ 2.68– 2.64 (m, 2H), 2.21– 2.16 (m, 2H), 1.62– 1.52 (m, 2H), 1.48– 1.38 (m, 9H), 1.36– 1.20 (m, 2H), 1.09 (m, 2H).
Step C: 6-(3-Bromo-2-bromomethyl-propionylamino)-hexanoic acid tert-butyl ester (95):
[0569] To a 50 mL round bottom flask equipped with a magnetic stirrer and nitrogen inlet was charged 6-aminohexanoic acid tert-butyl ester (94, 0.50 g; 2.7 mmol) and dimethylaminopyridine (0.03 g; 0.27 mmol; 0.10 equiv.) in DCM (5.0 mL). This solution was chilled to 0 oC via an ice bath. 3-Bromo-2-bromomethyl-propionyl chloride (92, 0.90 g; 3.4 mmol; 1.2 equiv.) was dissolved in DCM (5 mL) and slowly added to the reaction mixture at 0 oC. Stir and slowly warm to ambient temperature overnight. Dilute reaction mixture with ethyl acetate, wash the organic mixture with H2O, 5% NaHCO3 and brine. The organics were dried over Na2SO4, filtered, concentrated and purified on a silica gel column eluting with 0- 100% ethyl acetate in hexanes to afford 0.49 g (42% yield) as a clear yellow oil. 1H-NMR (300 MHz, CDCl3): ^ 5.92 (br s, 1H), 3.64– 3.58 (m, 2H), 3.54– 3.48 (m, 2H), 3.36– 3.29 (m, 2H), 2.89– 2.83 (m, 1H), 2.24– 2.20 (m, 2H), 1.65– 1.51 (m, 4H), 1.44– 1.35 (m, 11H).
Step D: 6-(3-Bromo-2-bromomethyl-propionylamino)-hexanoic acid (96):
[0570] To a 50 mL round bottom flask equipped with a magnetic stirrer and nitrogen inlet was charged 6-(3-Bromo-2-bromomethyl-propionylamino)-hexanoic acid tert-butyl ester (95, 0.26 g; 0.62 mmol) and trifluoroacetic acid (0.70 mL; 9.3 mmol; 15 equiv.) in DCM (10 mL). This solution was stirred at ambient temperature overnight, concentrated to dryness, dissolved in acetonitrile and H2O (1.0 mL each), frozen and lyophilized to afford 0.22 g (100%) as a solid. MS (ESI, pos.): calc’d for C10H17Br2NO3, 359.0 found 358.0, 360.0, 362.0 (M+H), 380.0, 382.0, 384.0 (M+Na), 356.0, 358.0, 360.0 (M-H). 1H-NMR (300 MHz, CDCl3): ^ 11.97 (s, 1H), 8.20– 8.16 (m, 1H), 3.58– 3.56 (d, 4H), 3.11– 3.04 (m, 2H), 3.02 - 2.97 (m, 1H), 2.21– 2.16 (m, 2H), 1.54– 1.37 (m, 4H), 1.33– 1.29 (m, 2H).
Step E: Boc-Val-Cit-4-aminobenzoic acid (97): 100 mg, 0.254 mmol) and di-tert-butyl dicarbonate (61 mg, 0.279 mmol), which were weighed into a round-bottom flask and treated with 1 M NaOH (2 mL) in THF (3 mL) and H2O (1.5 mL). The flask was sealed via rubber septum and the reaction was stirred at ambient temperature overnight, concentrated in vacuo and neutralized to pH 7 with dropwise addition of 1 M HCl (2 mL). The aqueous the reaction with ethyl acetate, dried the organic layer over Na2SO4, filter and concentrated to give an off-white solid (93 mg, 74%). MS (ESI, pos.): calc’d for C23H35N5O7, 493.5; found 394.2 (M+1-Boc), 494.2 (M+H), 516.2 (M+Na).
Step F: Maytansin-N-methyl-L-alanine-N-4-aminobenzamide-Cit-Val-Boc (98):
[0572] The title compound was prepared from the product of the preceding step Boc- Val-Cit-4-aminobenzoic acid (97, 76 mg, 0.154 mmol) and maytansin-N-methyl-L-alanine (9, 50 mg, 0.077 mmol) using the method from Step A of Example 5, to give a white solid (22 mg, 26%). MS (ESI, pos.): calc’d for C55H77ClN8O15,1124.5; found 1107.2 (M-H2O), 1125.3 (M+H), 1147.2 (M+Na).
Step G: Maytansin-N-methyl-L-alanine-N-4-aminobenzamide-Cit-Val (99):
[0573] The title compound was prepared from the product of the preceding step (98, 20 mg, 0.018 mmol) weighed into a round-bottom flask dissolved in ACN (3 mL) and H2O (1 mL) and treated with TFA (1 mL). The flask was sealed via rubber septum, purged with nitrogen, and the reaction was stirred at ambient temperature. After 48 h the reaction was purified directly on a 30 g C18 RediSep Gold Aq column via ISCO system (gradient elution: 10– 65% MeCN in water, 0.05% acetic acid in both, over 20 min). The product-containing fractions were combined, partially concentrated in vacuo, frozen on dry ice, and lyophilized to give an off-white solid (8 mg, 46%). MS (ESI, pos.): calc’d for C50H69ClN8O13, 1024.5; found 1007.2 (M-H2O), 1026.2 (M+H).
Step H: Maytansin-N-methyl-L-alanine-N-4-aminobenzamide-Cit-Val-capryl- bis(BrMe)acrylamide (100):
[0574] The title compound was prepared from the product of the preceding step (99, 8 mg, 0.008 mmol) and 6-(3-Bromo-2-bromomethyl-propionylamino)-hexanoic acid (96), using the method from Step A of Example 5, to give a white solid (8 mg, 75%). MS (ESI, CDCl3): δ 9.32 (s, 1H), 8.72 (m, 1H), 8.37 (m, 1H), 7.65 (m, 2H), 7.45 (m, 1H), 7.34 (m, 1H), 7.32 (m, 2H), 7.22-7.18 (m, 2H), 6.89-6.84 (m, 2H), 6.70-6.60 (m, 2H), 6.44 (m, 1H), 6.32 (br s, 1H), 6.12 (s, 1H), 5.76-5.71 (m, 1H), 5.65 (s, 1H), 5.37-5.29 (m, 3H), 4.86-4.70 (m, 3H), 4.52 (br s, 1H), 4.31-4.25 (m, 1H), 4.12 (m, 1H), 3.99 (s, 3H), 3.65-3.58 (m, 1H), 3.51 (m, 1H), 3.34-3.21 (m, 8H), 3.13-3.02 (m, 5H), 2.88 (s, 3H), 2.68 (m, 1H), 2.28-2.17 (m, 3H), 2.11–1.83 (m, 2H), 1.80-1.70 (m, 2H), 1.66 (s, 3H), 1.57-1.49 (m, 4H), 1.47-1.43 (m, 3H), 1.30-1.26 (m,7H), 1.00-0.91 (m, 6H), 0.85 (s, 3H).
EXAMPLE 37
Conjugate Preparation and Characterization
[0575] Five antibodies were conjugated to the linker-payload compounds of the disclosure using the procedures below. The four targeting antibodies used in these experiments were: (1) a PSMA antibody having the heavy and light chain variable domains of clone AB-PG1-XG1-006 as set forth in WO2007002222A2, (2) anti-MUC16 antibody having variable regions derived from clone 3A5 from WO2007001851, and (3) two PRLR antibodies having the heavy and light chain variable domains of clone H1H6765P and H1H6958N2 as set forth in WO2015026907A1. All the monoclonal antibodies were expressed in CHO cells and purified by Protein A. A non-binding isotype control derived from an antigen having no relation to oncology was also used. EXAMPLE 38 Conjugation Method for Compound 10
Conjugation Method for Maleimides
[0576] The antibody (1-10 mg/ml) in 50 mM HEPES, 150 mM NaCl, pH 7.5, was treated with 1 mM dithiothreitol at 37 °C for 30 min. After gel filtration (G-25, pH 4.5 sodium acetate), the maleimido linker payload derivative 10, 15, 20, 25, 60, and 78 (1.2 equivalents/SH group) in
DMSO (10 mg/ml) was added to the reduced antibody and the mixture adjusted to pH 7.0 with 1M HEPES (pH 7.4). After 1 h the reaction was quenched with excess N-ethyl maleimide. The conjugates were purified by size exclusion chromatography and sterile filtered. Protein concentrations and payload to antibody ratios were determined by UV monomeric, and RP-HPLC established that there was <0.5% unconjugated linker payload.
All conjugated antibodies were analyzed by UV for linker payload loading values according to Hamblett et al, Cancer Res 2004107063. Yields and payload to antibody ratios are reported in Table 1.
EXAMPLE 39
Conjugation Method for Active Esters [0577] The antibodies (1-10 mg/ml) in 50 mM HEPES, 150 mM NaCl, pH 8.0, and 15% (v/v) DMA were conjugated with a 6 fold excess of compound 77 for 1-2 hours at ambient temperature. The conjugates were purified by size exclusion chromatography and sterile filtered. Protein and linker payload concentrations were determined by UV spectral analysis. Size-exclusion HPLC established that all conjugates used were >95% monomeric, and RP- HPLC established that there was <0.5% unconjugated linker payload. Yields are reported in Table 1 based on protein. All conjugated antibodies were analyzed by UV for linker payload loading values according to Hamblett et al, Cancer Res 2004107063. Yields and payload to antibody ratios are reported in Table 1.
Figure imgf000267_0001
Figure imgf000268_0001
In Vitro Linker-Payload Cell-free Enzymatic Assays
Cathepsin B incubation
[0578] In vitro cell-free enzymatic assay procedure was adopted from Dubowchik, et al. Bioconjugate Chem.200213855. The linker payload 10 was set at 100 μg/mL final in 25 mM sodium acetate buffer, 1 mM EDTA, pH 5.0 and pre-incubated at 37˚C. Cathepsin B mM DTT, 15 mM EDTA to 2 equivalents of cathepsin B stock. The activated cathepsin B solution was added to the substrate solutions at a 1:20 molar ratio (purified H2O, instead of activated cathepsin B was added for the control sample.) Samples were incubated at 37˚C overnight and the resulting samples are detected by LC-MS through Q1 Scan.
LC-MS detection
[0579] Samples are centrifuged at 12,000 g for 5 min. Supernatant was recovered and analyzed by liquid chromatography-mass spectrometry (Thermo Quantiva) by combined infusion of 0.3 ml/min of 30:70 mobile phase B:A (Mobile Phase A: 0.1%FA in H2O; Mobile Phase B: 0.1% FA in Acetonitrile) at 20 μl/min from supernatant. MS1 is set at an appropriate range for detection of molecular ion of either linker payload or payload. The supernatant contained the predicted payload, p-amino-benzamide maytansinoid (27), with a mass of 791.27 M+Na (calc’d monoisotopic mass for C39H49ClN4O10, 768.31) and the control samples without cathepsin B contained 10 with a mass of 1240.50 M+Na (calc’d
monoisotopic mass for C60H80ClN9O16, 1217.54). No predicted payload molecular ion was detected in the control samples.
[0580] The results of this Example are significant in part because cathepsin B proteolysis of 10 should only occur after internalization of the ADC in the cell where the enzyme exists. Off target effects should be reduced since the antibody delivers the cytotoxic payload directly to targeted cells.
EXAMPLE 41 In Vitro Cytotoxicity Assays [0581] In this Example, the ability of various antibody-drug conjugates or their associated payloads to kill antigen-expressing tumor cells in vitro was assessed.
[0582] Ovcar3 (Muc16+) or C4-2 (PSMA+) cells were seeded in 96 well plates at 3000 (C42) cells per well in complete growth media and grown overnight. For cell viability curves, serially diluted conjugates or payloads were added to the cells at final concentrations ranging from 300 nM to 5 pM and incubated for 3 days. To measure viability, cells were incubated with CCK8 (Dojindo) for the final 1-3 hours and the absorbance at 450nm (OD450) was determined on a Victor (Perkin Elmer). Background OD450 levels determined from digitonin (40 nM) treated cells were subtracted from all wells and viability is expressed as a percentage of the untreated controls. IC50 values were determined from a four-parameter IC50 values are corrected for payload equivalents.
[0583] In C4-2 cells (prostate cancer line), natively expressing PSMA at 271 fold above isotype control binding, the maytansinoid conjugates PSMA-10 and PSMA-25 possessed IC50 values of 0.11 and 0.59 nM, respectively (FIGs.2-5). The payloads 27 and 33 alone had IC50 values of 0.20 and 0.55 nM, respectively. The naked PSMA antibody and isotype control were devoid of any anti-proliferation activity at the concentrations assayed.
[0584] In Ovcar-3 cells (ovarian cancer line), natively expressing MUC16 at 320 fold above isotype control binding, the maytansinoid conjugates MUC16-10 and MUC16-25 possessed IC50 value of 0.74 and 0.63 nM, respectively (FIGs.2-5). The payloads 27 and 33 alone had IC50 values of 0.06 and 0.11 nM, respectively. The naked MUC16 antibody and isotype control were devoid of any anti-proliferation activity at the concentrations assayed.
[0585] Table 2 lists the anti-proliferating ability of the payloads only in both Ovcar3 (Muc16+) or C4-2 (PSMA+) cells. All compounds possess sub-nanomolar activities with compounds 35 and 37 at or near single digit picomolar IC50s. Table 2
Figure imgf000270_0001
[0586] Assay/Experiment Type: Cloning, expression and purification of antibodies modified to contain site-specific conjugation motifs [0587] This Example provides the generation of antibodies with amino acids sequences that allow site-specific conjugation by transglutaminase reactions.
[0588] To generate antibodies, mutagenesis was performed on a plasmid encoding the CH1, CH2, and CH3 domains of human IgG1 (amino acids 1 through 330 of UniprotKB accession no. P01857) to generate an N to Q mutation at position 180 using a QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent, # 210516). Two antibody variable region heavy (VH) fragments, one encoding the VH of an anti-human PRLR antibody, H1H6958N2 (international patent application WO 2015026907 A1) and another encoding the VH of the isotype control antibody recognizing an exogenous antigen, were selected. Primers were designed (idtdna.com) to amplify the VH regions of these two antibodies using Kapa HiFi DNA polymerase (Kapa Biosciences; # KK2102). While PCR amplification was proceeding, a plasmid containing the human IgG1 N180Q mutation was digested with LguI enzyme (Fermentas, # FD1934) at 37°C for 30 minutes. Once the amplification was complete, the digested human IgG1 plasmid and both PCR products were run out on a 1% agarose gel containing SYBR Safe stain (Life Technologies, # S33102). Bands of the appropriate size were identified and excised from the gel using a clean razor blade. All excised products were purified using a Gel Extraction kit (Qiagen, #28704). An In-Fusion cloning reaction (Clontech, # 638911) was then performed using a ratio of 3:1 of digested human IgG1 vector to VH PCR product and then incubated for 15 minutes at 50°C. After incubation, each reaction was transformed into Mix and Go competent cells (Zymo, # T3007), incubated on ice for 5 minutes, and competent cells were plated on LB +
Carbenicillin plates (Teknova, VWR, # 101320-126), which were incubated overnight at 37°C.
[0589] The following day, single colonies were inoculated from the plate into LB broth containing 100 μg/mL Carbenicillin and grown overnight shaking in a table top incubator at 37°C. Cells were then pelleted by centrifugation and miniprepped on a Hamilton Starlet robot using a PureLink HiPure Plasmid miniprep kit (Thermo Fisher, #K210003). Purified DNA was sequenced and results were analyzed using Sequencher software
(GeneCodes). A clone from each ligation reaction was selected for re-transformation into on LB plates containing Carbenicillin, which were incubated overnight at 37°C.
[0590] The following day, a single colony was picked from each plate and grown in LB broth containing 100 μg/mL Carbenicillin for 3 to 4 hours. The sample was then diluted into LB broth containing 100 μg/ml Carbenicillin and transferred to a 37°C shaking incubator to grow overnight. A maxiprep DNA extraction was then performed on both plasmids and the full DNA open reading frames were sequenced. Once the sequences were confirmed, the heavy chain DNA along with previously cloned affiliated light chain DNA was stably transfected into a CHO cell line to produce each antibody.
[0591] The H1H6958N2 (international patent application WO 2015026907 A1) containing the N180Q Fc mutation is referred to as PRLR-Q, and the isotype control antibody recognizing an exogenous antigen also containing the N180Q Fc mutation is referred to as ISOTYPE CONTROL-Q.
Example 43
[0592] Bacterial Transglutaminase Conjugation
[0593] PRLR-Q (MW 145438 Da) and Isotype Control-Q (MW 144602 Da) antibodies were conjugated at 1-10 mg/mL in PBS pH 7.4. Linker payload 63 was added in a 10 to 25-fold molar excess over antibody and the enzymatic reaction was initiated by addition of 1-5 units of bacterial transglutaminase (Zedira, T1001 ) per mg antibody and incubated with shaking at 37°C for 4-16 hours. The conjugates were purified by size exclusion chromatography and sterile filtered. Protein and linker payload concentrations were determined by UV spectral analysis. Size-exclusion HPLC established that all conjugates used were >95% monomeric. Yields are reported in Table 1 based on protein. All conjugated antibodies were analyzed by UV for linker payload loading values according to Hamblett et al, Cancer Res 2004107063. In addition, the conjugates were analyzed by ESI-MS for linker payload loadings using a Waters Synapt G2-Si QTOF mass spectrometry coupled with Acquity UPLC. The chromatographic separation was achieved on a C4 column (Waters protein BEH C4, 50 mm X 1 mm, 1.7 μm) in a 25 minute gradient (minute:percentage of mobile pahse B; 0:20%, 1:20%, 18:40%, 18.1:90, 20:95%, 20.8:95%, 20.9:20% 25:20%). The mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic m/z 700-5000 for 25 minutes with major parameters as listed (Capillary voltage 3.2 kV; Sampling Cone 150; Source Offset at 80; Source temperatures 120 oC; Desolvation temperature 500 oC; Trap collision Energy 30; Transfer Collision Energy Off; Gas controls OFF; Resolving Quadrupole: LM resolution at 4.7). The combined spectra were
deconvoluted with MaxEnt function within MassLynx software. The resulting molecular ions which when weighted according to intensities corresponded to the loadings listed in Table 3. The actual mass spec spectra are listed in Figures 42 and 43. [0594] Table 3. The summary of intensity-weighted average linker-payload loadings in PRLR-Q and Isotype Control-Q conjugates for compound 63. PRLR‐Q‐63  Isotype Control‐Q‐63 
Molecular  Corresponding  Relative  Intensity  Molecular  Corresponding  Relative  Intensity 
Figure imgf000273_0001
Example 44
[0595] Equilibrium dissociation constants (KD values) for human PRLR binding to purified anti-PRLR antibodies that were either unmodified H1H6958N2, PRLR-Q, and PRLR-Q-63 were determined using a real-time surface plasmon resonance biosensor using a Biacore 3000 instrument. The Biacore sensor surface was first derivatized by amine coupling with a monoclonal mouse anti-human Fc antibody (GE, # BR-1008-39) to capture anti-PRLR monoclonal antibodies. All binding studies were performed in 0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, and 0.05% v/v Surfactant Tween-20 (HBS-ET running buffer) at 25°C and 37°C. Different concentrations of human PRLR extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (hPRLR-MMH; SEQ ID NO: 401 as described in patent application WO 2015026907 A1), in HBS-ET running buffer (ranging from 40nM to 3.33nM) were injected over the anti-PRLR antibody captured surface for 4 minutes at a flow rate of 50µL/minute and their dissociation in HBS-ET running buffer was monitored for 8 minu ssociation rate constant (ka) and dissociation rate constant (kd) were
Figure imgf000273_0002
curve fitting software. Binding dissociation equilibrium constants (KD) and dissociative half- lives (t½) were calculated from the kinetic rate constants as:
[0596] Binding
Figure imgf000274_0001
kinetic parameters for hPRLR-MMH binding to different anti-PRLR antibodies at 25°C are shown in Table 4. At 25°C, hPRLR-MMH bound to the parental antibody H1H6958N2 with a KD value of 1.09nM. Human PRLR-MMH bound to the PRLR- Q with a KD value of 850pM and to PRLR-Q-63 with a KD value of 1.50nM.
Table 4: Binding Kinetics parameters of anti-PRLR antibodies binding to hPRLR-MMH at 25°C. Antibody mAb 100nM ka kd KD t½
Figure imgf000275_0001
Example 45
[0597] The present example provides cytotoxicity assays for conjugates provided herein. To evaluate the ability of anti-PRLR antibodies conjugated with 63, 60, 77, and 78 to kill a PRLR expressing cell line, an in vitro cytotoxicity assay using a T47D ductal carcinoma line (ATCC, # HTB-133), which was previously determined to express >27,000 copies of human PRLR at its cell surface, was utilized.
[0598] For the assay, T47D cells were seeded onto white 96 well plates at 2,000 cells/ well in media containing DMEM supplemented with 10% FBS, NEAA, and
pencillin/streptomycinin (complete media). They were grown overnight at 37°C in 5% CO2. To determine cell viability curves, the following day antibody drug conjugates, unconjugated antibodies, or free payloads were added to the cells at final serial dilutions ranging from 100 nM to 0.01 nM in complete medium and then incubated for an additional 5 days. Luciferase activity was detected after the addition of CellTiter-GloTM reagent (Promega, G7571) to each well, which contains reagents to lyse the remaining viable cells to release their ATP, ATPase inhibitors to prevent degradation of the ATP, as well as luciferin and luciferase to catalyse the luminescent reaction. Viable cells prior to addition of CellTiter-Glo will be the only source of ATP since the dead cells in culture will not synthesis ATP and any of their released ATP will be destroyed via endogenous ATPases. Relative light units (RLUs) were measured on a Victor luminometer (PerkinElmer) and the results were determined using a four- parameter logistic equation over a 10-point response curve (GraphPad Prism). All measured values and calculated IC50 values were corrected for payload equivalents. All IC50 values are expressed results are summarized in FIGS.45 through 48.
[0599] As shown in FIG.44, the anti-PRLR antibody site-specifically conjugated with 63 (PRLR-Q-63) demonstrated cytotoxicity of T47D cells with an IC50 of 1.1 nM and a maximum percent killing of 55%. The free payload, 29, demonstrated cytotoxicity of T47D cells with an IC50 of 0.07nM and a maximum percent killing of 67%. An isotype control antibody conjugated with 63 (ISOTYPE CONTROL-Q-63) did not demonstrate any killing of T47D cells, and the unconjugated anti-PRLR antibody (PRLR-Q) did not demonstrate any killing of T47D cells.
[0600] As shown in FIG.45, the anti-PRLR antibody conjugated with 60 (PRLR-60) demonstrated cytotoxicity of T47D cells with an IC50 of 0.6 nM and a maximum percent killing of 60%. The free payload, 49, demonstrated cytotoxicity of T47D cells with an IC50 of 0.03 nM and a maximum percent killing of 69%. An isotype control antibody conjugated with 60 (ISOTYPE CONTROL-60) did not demonstrate any killing of T47D cells, and the unconjugated anti-PRLR antibody (PRLR) did not demonstrate any killing of T47D cells.
[0601] As shown in FIG.46, the anti-PRLR antibody conjugated with 77 (PRLR-77) demonstrated cytotoxicity of T47D cells with an IC50 of 0.4 nM and a maximum percent killing of 69%. The free payload, 27, demonstrated cytotoxicity of T47D cells with an IC50 of 0.1 nM and a maximum percent killing of 67%. An isotype control antibody conjugated with 77 (ISOTYPE CONTROL-77) did not demonstrate any killing of T47D cells, and the unconjugated anti-PRLR antibody (PRLR) did not demonstrate any killing of T47D cells.
[0602] As shown in FIG.47, the anti-PRLR antibody conjugated with 78 (PRLR-78) demonstrated cytotoxicity of T47D cells with an IC50 of 0.9 nM and a maximum percent killing of 61%. The free payload, 27, demonstrated cytotoxicity of T47D cells with an IC50 of 0.1 nM and a maximum percent killing of 67%. An isotype control antibody conjugated with 78 (ISOTYPE CONTROL-78) did not demonstrate any killing of T47D cells, and the unconjugated anti-PRLR antibody (PRLR) did not demonstrate any killing of T47D cells. [0603] Table 5 lists the anti-proliferating ability of the payloads only in Ovcar3 (Muc16+), C4-2 (PSMA+), and T47D (PRLR+) cells.
Table 5 ^ C4‐2 Ovcar3 T47D
Figure imgf000276_0001
[0
Figure imgf000277_0001
ly illustrative and non-limiting. Those skilled in the art will recognize or will be able to ascertain using no more than routine experimentation, numerous equivalents of specific compounds, materials and procedures. All such equivalents are considered to be within the scope and are encompassed by the appended claims.

Claims

What is claimed is: 1. A compound of Formula (I):
Figure imgf000278_0001
or a pharmaceutically acceptable salt thereof,
wherein:
A is arylene or heteroarylene;
L is a linker;
BA is a binding agent; and
k is an integer from 1 to 30. 2. The compound of claim 1, wherein A is:
wherein:
Figure imgf000278_0002
R1, independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkyl, haloalkoxy, heteroaryl, heterocycloalkyl, hydroxyl, cyano, nitro, , , , or azido,
Figure imgf000278_0003
g ;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5.
4. The compound of any of
Figure imgf000279_0001
wherein A is:
wherein n is 0, 1 or 2.
Figure imgf000279_0002
5. The compound of any of claims 1-4, wherein n is 0. 6. The compound of any of claims 1-4, wherein R1 is, independently at each occurrence, alkoxy or halo. 7. The compound of any of claims 1-6, wherein L is:
wherein:
Figure imgf000279_0003
SP is a spacer; is one or more bonds to the binding agent;
A1 is an amino acid; and
Figure imgf000279_0004
AA2 is an amino acid.
8. The compound of any of claim 7, wherein AA1-AA2 is: valine-citrulline, citrulline-valine, lysine-phenylalanine, phenylalanine-lysine, valine-asparagine, asparagine- valine, threonine-asparagine, asparagine-threonine, serine-asparagine, asparagine-serine, phenylalanine-asparagine, asparagine-phenylalanine, leucine-asparagine, asparagine-leucine, isoleucine-asparagine, asparagine-isoleucine, glycine-asparagine, asparagine-glycine, glutamic acid-asparagine, asparagine-glutamic acid, citrulline-asparagine, asparagine- citrulline, alanine-asparagine, or asparagine-alanine. 9. The compound of any of claims 1-6, wherein L is: wherein:
Figure imgf000280_0001
SP is a spacer; is one or more bonds to the binding agent;
AA1
Figure imgf000280_0004
is an amino acid side chain; and
RAA2 is an amino acid side chain.
10. The compound of claim 9, wherein L is:
wherein:
Figure imgf000280_0002
SP is a spacer; and is one or more bonds to the binding agent.
11.
Figure imgf000280_0005
he compound of any of claims 7-10, wherein SP comprises a C5-7 alkylene. 12. The compound of claim 11, wherein SP is:
wherein:
Figure imgf000280_0003
is a bond to the binding agent; and
b is an integer from 2 to 8.
Figure imgf000280_0006
13. The compound of any of claims 1-6, wherein L is:
Figure imgf000281_0001
is a bond to the binding agent; and
Figure imgf000281_0003
is an integer from 2 to 8.
14. The compound of any of claims 1-6, wherein L is:
r
Figure imgf000281_0002
wherein:
BA is an antibody;
RN is a hydrogen atom or alkyl;
RM is alkyl; the two bonds represented by are bonds to cysteines of the antibody; and b is an integer from 2 to 8.
15. The compound of claim 13, wherein L is: wherei
Figure imgf000282_0001
16. The compound of any of claims 1-6, wherein L is:
or
Figure imgf000282_0002
BA is an antibody and
L is:
where
Figure imgf000283_0001
in the two bonds represented by are bonds to cysteines of the antibody. 18. The compound of any of cla
Figure imgf000283_0003
ims 1-6, wherein L is:
;
Figure imgf000283_0002
wherein is a bond to the binding agent.
19. The compound of claim 1, wherein:
A is: , wherein:
Figure imgf000284_0001
R1, independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkyl, haloalkoxy, heteroaryl, heterocycloalkyl, cyano, nitro, , , , or azido,
whe
Figure imgf000284_0004
n is an integer from 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5; and
L i
Figure imgf000284_0002
SP is a spacer; is one or more bonds to the binding agent;
Figure imgf000284_0005
RAA1 is an amino acid side chain; and
RAA2 is an amino acid side chain. 20. The compound of claim 1, wherein: A is:
;
Figure imgf000284_0003
wherein R1 is, independently at eac occurrence, alkyl, alkoxy, haloalkyl, or halo; and L is: wherein:
Figure imgf000285_0001
is a bond to the binding agent; and
Figure imgf000285_0003
is an integer from 2 to 8.
21. The compound of claim 1, wherein: A is:
wherein:
Figure imgf000285_0004
n is 0, 1 or 2; and
R1 is, independently at each occurrence, alkyl, alkoxy, haloalkyl, or halo L is:
wherein:
Figure imgf000285_0002
is a bond to the binding agent. 22.
Figure imgf000285_0005
The compound of claim 1, wherein the compound is:
Figure imgf000286_0001
,
,
Figure imgf000287_0001
Figure imgf000288_0001
,
,
Figure imgf000289_0001
,
,
Figure imgf000290_0001
,
Figure imgf000291_0001
,
Figure imgf000291_0002
Figure imgf000292_0001
,
,
Figure imgf000293_0001
OC 3 3
Figure imgf000294_0001
,
, ,
Figure imgf000295_0001
N
,
Figure imgf000296_0001
,
Figure imgf000296_0002
,
, , ,
Figure imgf000297_0001
,
Figure imgf000298_0001
,
,
Figure imgf000299_0001
,
, ,
Figure imgf000300_0001
,
,
Figure imgf000301_0001
Figure imgf000302_0001
Figure imgf000303_0001
Figure imgf000304_0001
Figure imgf000305_0001
,
Figure imgf000306_0001
Figure imgf000307_0001
Figure imgf000308_0001
Figure imgf000309_0001
Figure imgf000310_0001
Figure imgf000311_0001
,
,
Figure imgf000312_0001
Figure imgf000313_0001
Figure imgf000314_0001
,
Figure imgf000315_0001
Figure imgf000316_0001
Figure imgf000317_0001
,
,
Figure imgf000318_0001
Figure imgf000319_0001
Figure imgf000320_0001
Figure imgf000321_0001
Figure imgf000322_0001
Figure imgf000323_0001
Figure imgf000324_0001
Figure imgf000325_0001
,
, , ,
Figure imgf000326_0001
Figure imgf000327_0001
Figure imgf000328_0001
Figure imgf000329_0001
Figure imgf000330_0001
;
Figure imgf000331_0001
Ab is an antibody;
is a bond to a cysteine of the antibody;
is a bond to a lysine of the antibody;
Figure imgf000331_0003
k is an integer from 1 to 30; and
t is an integer from 1 to 8.
23. The compound of claim 22, wherein the compound is:
.
Figure imgf000331_0002
. e compoun o any o cams - , weren ts .
26. The compound of any of claims 1-25, wherein the antibody is an anti-MUC16 antibody or anti-PSMA antibody. 27. A compound of Formula II:
Figure imgf000332_0001
or a pharmaceutically acceptable salt thereof,
wherein A is arylene or heteroarylene. 28. The compound of claim 27, wherein: A is:
wherein:
Figure imgf000332_0002
R1 is, independently at each occurrence, alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkyl, haloalkoxy, heteroaryl, heterocycloalkyl, cyano, nitro, , or azido,
Figure imgf000332_0003
n is an integer from 0 to 4;
m is and integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5. compound of the Formula (IIA):
Figure imgf000333_0001
30. The compound of claim 28, wherein the compound of Formula (II) is a compound of the Formula (IIB):
Figure imgf000333_0002
31. The compound of claim 28, wherein the compound of Formula (II) is a compound of the Formula (IIB2):
Figure imgf000333_0003
32. The compound of claim 28, wherein the compound of Formula (II) is a compound of the Formula (IIB3):
Figure imgf000334_0001
33. The compound of any of claims 28-32, wherein R1 is, independently, C1-6 alkoxy, haloalkyl or halo. 34. The compound of any of claims 28-33, wherein R1 is, independently, alkoxy or halo. 35. The compound of any of claims 28-34, wherein n, m, p, and q are 0, 1 or 2. 36. The compound of any of claims 28-35, wherein n, m, p, and q are 0. 37. The compound of claim 27, wherein the compound is:
,
Figure imgf000334_0002
,
Figure imgf000335_0001
,or
Figure imgf000336_0001
pendently at each occurrence, alkoxy, haloalkyl, or halo. 39. The compound of claim 38, wherein R1 is alkoxy. 40. The compound of claim 38, wherein R1 is methoxy, trifluoromethyl, chloro, or fluoro. 41. The compound of claim 40, wherein R1 is methoxy. 42. The compound of claim 40, wherein the compound is:
,
Figure imgf000336_0002
,
, , , , or
Figure imgf000337_0001
.
43.
Figure imgf000338_0001
f claims 1- 42 and a pharmaceutically acceptable excipient 44. A method of treating a proliferative disorder comprising administering to a patient having said disorder a therapeutically effective amount of a compound of any of claims 1-42 or a composition of claim 43. 45. A compound of Formula P1:
Figure imgf000338_0002
wherein A is arylene or heteroarylene and RL is a reactive linker. 46. The compound of claim 45, wherein the compound of Formula P1 is a compound of Formula P1B:
Figure imgf000338_0003
wherein
A is:
Figure imgf000339_0001
R is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkyl, haloalkoxy, heteroaryl, heterocycloalkyl, cyano, nitro, , , or azido,
Figure imgf000339_0002
n is an integer from 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5; and
SPR is a reactive spacer.
47. The compound of claim 45, wherein the compound of Formula P1 is a compound of Formula P1H:
where
Figure imgf000339_0003
in:
R1 is hydrogen atom, alkoxy, halo, haloalkyl, or trifluoromethyl; and b is an integer from 2 to 8. 48. The compound of claim 47, wherein R1 is alkoxy or haloalkyl.
50. The compound of claim 48, wherein R1 is methoxy. 51. The compound of claim 45, wherein the compound of Formula P1 is a compound of Formula P1I: H CH OH OCH 3 3 O N 3
whe
Figure imgf000340_0001
R1 is a hydrogen atom, alkoxy, halo, haloalkyl, or trifluoromethyl; and b is an integer from 2 to 8. 52. The compound of claim 45, wherein the compound of Formula P1 is a compound of Formula P1J:
Figure imgf000340_0002
wherein:
R1 is a hydrogen atom, alkoxy, halo, haloalkyl, or trifluoromethyl; and b is an integer from 2 to 8. compound of Formula P1N or P1U:
Figure imgf000341_0001
wherein RN is a hydrogen atom or alkyl, RM is alkyl, R1 is a hydrogen atom, alkoxy, halo, haloalkyl, or trifluoromethyl, and b is an integer from 2 to 8. 54. The compound of claim 45, wherein the compound of Formula P1 is a compound of Formula P1P or Formula P1T:
Figure imgf000342_0001
wherein RN is a hydrogen atom or alkyl, RM is alkyl, R1 is a hydrogen atom, alkoxy, halo, haloalkyl, or trifluoromethyl, and b is an integer from 2 to 8. 55. The compound of claim 45, wherein the compound of Formula P1 is a compound of Formula P1Q:
w teger
Figure imgf000342_0002
from 2 to 8. 56. The compound of claim 45, wherein the compound of Formula P1 is a compound of Formula P1V or Formula P1W:
Figure imgf000343_0001
Figure imgf000343_0002
wherein R1 is a hydrogen atom, alkoxy, halo, haloalkyl, or trifluoromethyl, g is an integer from 2 to 8, and b is an integer from 2 to 8. 57. The compound of claim 45, wherein the compound of Formula P1 is a compound of Formula P1K:
Figure imgf000343_0003
wherein R1 is a hydrogen atom, alkoxy, halo, haloalkyl, or trifluoromethyl.
58. The compound of claim 45, wherein the compound is: ,
, ,
Figure imgf000344_0001
,
, , ,
Figure imgf000345_0001
,
,
Figure imgf000346_0001
,
Figure imgf000347_0001
3
,
Figure imgf000348_0001
,
Figure imgf000349_0001
, ,
Figure imgf000349_0002
,
, , ,
Figure imgf000350_0001
,
,
Figure imgf000351_0001
,
Figure imgf000352_0001
O
3 ,
Figure imgf000353_0001
,
,
Figure imgf000354_0001
Figure imgf000355_0001
,
,
Figure imgf000356_0001
,
Figure imgf000357_0001
,
Figure imgf000358_0001
Figure imgf000358_0002
Figure imgf000359_0001
Figure imgf000360_0001
Figure imgf000361_0001
Figure imgf000362_0001
,
,
Figure imgf000363_0001
Figure imgf000364_0001
,
, ,
,
Figure imgf000365_0001
, , , ,
Figure imgf000366_0001
,
Figure imgf000367_0001
,
Figure imgf000368_0001
,
Figure imgf000368_0002
,
Figure imgf000368_0003
Figure imgf000369_0001
,
, ,
,
Figure imgf000370_0001
, , , ,
Figure imgf000371_0001
,
Figure imgf000372_0001
,
,
Figure imgf000373_0001
,
, , ,
Figure imgf000374_0001
,
, ,
Figure imgf000375_0001
, , , ,
Figure imgf000376_0001
3
,
Figure imgf000377_0001
,
Figure imgf000378_0001
, ,
Figure imgf000378_0002
,
, , ,
Figure imgf000379_0001
,
, , ,
Figure imgf000380_0001
O N
Figure imgf000381_0001
,
3 ,
Figure imgf000382_0001
,
,
Figure imgf000383_0001
,
, , ,
Figure imgf000384_0001
Figure imgf000385_0001
, , , ,
Figure imgf000386_0001
,
Figure imgf000387_0001
,
,
Figure imgf000388_0001
Figure imgf000389_0001
,
Figure imgf000390_0001
, , , ,
Figure imgf000391_0001
,
Figure imgf000392_0001
,
,
Figure imgf000393_0001
,
,
Figure imgf000394_0001
Figure imgf000395_0001
, , , ,
Figure imgf000396_0001
,
Figure imgf000397_0001
,
Figure imgf000397_0002
,
, , ,
Figure imgf000398_0001
Figure imgf000399_0001
,
, , ,
Figure imgf000400_0001
Figure imgf000401_0001
,
Figure imgf000402_0001
.
Figure imgf000403_0001
the compound is: , ,
Figure imgf000403_0002
. 60.
Figure imgf000404_0001
Figure imgf000404_0002
Figure imgf000405_0001
with a binding agent under conjugation conditions, wherein A is arylene or heteroarylene;
L is a linker;
BA is a binding agent;
k is an integer from 1 to 10;
and RL is a reactive linker.
61. The method of claim 60, wherein the compound of Formula P1 is prepared by contacting a compound of Formula P2:
Figure imgf000405_0002
with a compound of Formul
under amide synthesis conditions
Figure imgf000405_0003
wherein RL is a reactive linker and A is arylene or heteroarylene.
62. A compound of Formula PP5:
Figure imgf000406_0001
or a salt thereof, wherein A is arylene or heteroarylene. 63. The compound of claim 62, wherein the compound is a compound of Formula PP5A:
Figure imgf000406_0002
wherein:
R1, independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, haloalkyl, haloalkoxy, heteroalkyl, aryl, alkaryl, aralkyl, halo, heteroaryl, heterocycloalkyl, cyano, nitro, , or azido,
Figure imgf000406_0003
s alkyl;
n is an integer from 0 to 4.
from
65. red by
Figure imgf000407_0001
contacting a compound of Formula PP3:
Figure imgf000407_0002
with a compound of Formula PP7:
Figure imgf000407_0003
wherein SPR is a reactive linker;
RAA1 is an amino acid side chain;
RAA1 is an amino acid side chain; and
A is arylene or heteroarylene.
66. The method of claim 65, wherein the compound of Formula PP3 is prepared by contacting a compound of Formula PP5 with a reducing agent:
Figure imgf000408_0001
67. The method of claim 66, wherein the reducing agent is zinc dust.
68. A method of making a compound of formula PP3:
Figure imgf000408_0002
, comprising:
(a) contacting a compound of formula P2
Figure imgf000408_0003
,
with a compound of formula PP6:
Figure imgf000409_0001
under amide synthesis conditions to form a compound of formula PP5:
Figure imgf000409_0002
wherein A is arylene or heteroarylene; and
(b) reducing the compound of formula PP5 by contacting the compound of formula PP5 with a reducing agent to form a compound of formula PP3.
69. The method of claim 68, wherein the reducing agent is zinc.
70. The method of claim 69, wherein the zinc is zinc dust.
71. The method of claim 68, wherein A is:
wherein:
Figure imgf000409_0003
R1, independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkyl, haloalkoxy, heteroaryl, heterocycloalkyl, hydroxyl, cyano, nitro, , , , or azido,
eteroalkyl;
Figure imgf000409_0004
n is an integer from 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5.
72. The method of claim 71, wherein A is:
73. The method of claim 71- A is:
Figure imgf000410_0001
wherein n is 0, 1 or 2.
Figure imgf000410_0002
74. The method of claim 71-73, wherein n is 0. 75. The method of claim 71-73, wherein R1 is, independently at each occurrence, alkoxy or halo. 76. The method of claim 71-73, wherein: A is:
wherein:
Figure imgf000410_0003
n is 0, 1 or 2; and
R1 is, independently at each occurrence, alkyl, alkoxy, haloalkyl, or halo. 77. The method of claim 71-73, wherein A is:
wherein n is 0 or 1; and R1 is alkoxy, h
Figure imgf000410_0004
l.
78. The method of claim 71-73, wherein A is:
Figure imgf000410_0005
,
wherein n is 0 or 1; and R1 is C1-6 alko
Figure imgf000411_0001
1-6 haloalkyl.
80. The method of any one of claims 68-79, wherein the reducing the compound of formula PP5 to form a compound of formula PP3 comprises reducing in an acid. 81. The method of claim 80, wherein the acid is selected from the group consisting of acetic acid, formic acid, pTsOH, and NH4Cl. 82. A compound having the following formula:
,
Figure imgf000411_0002
w ere n s ary ene or eteroary ene,
L1 and L2 are linkers,
BA is a binding agent,
k is an integer from 0 to 30, and
t is an integer from 0 to 8.
83. The compound of claim 82, wherein the sum of k and t equals an integer from 1 to 8. 84. The compound of claim 82, wherein A is:
,
wherein:
Figure imgf000411_0003
aralkyl, halo, haloalkyl, haloalkoxy, heteroaryl, heterocycloalkyl, cyano, nitro, , or azido,
Figure imgf000412_0001
;
n is an integer from 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and
q is an integer from 0 to 5.
85. The compound of any of claims 82-84, wherein L1 or L2 are:
wherein:
Figure imgf000412_0003
SP is a spacer; is one or more bonds to the binding agent;
Figure imgf000412_0004
AA1 is an amino acid; and
AA2 is an amino acid.
86. The compound of claim 84, wherein AA1-AA2 is: valine-citrulline, citrulline- valine, lysine-phenylalanine, phenylalanine-lysine, valine-asparagine, asparagine-valine, threonine-asparagine, asparagine-threonine, serine-asparagine, asparagine-serine, phenylalanine-asparagine, asparagine-phenylalanine, leucine-asparagine, asparagine-leucine, isoleucine-asparagine, asparagine-isoleucine, glycine-asparagine, asparagine-glycine, glutamic acid-asparagine, asparagine-glutamic acid, citrulline-asparagine, asparagine- citrulline, alanine-asparagine, or asparagine-alanine. 87. The compound of any one of claims 82-86, wherein L1 or L2 are:
wherein:
Figure imgf000412_0002
SP is a spacer; is one or more bonds to the binding agent; RAA2 is an amino acid side chain.
88. The compound of claim 87, wherein SP is:
wherein:
Figure imgf000413_0001
is a bond to the binding agent; and
is an integer from 2 to 8.
Figure imgf000413_0003
89. The compound of claim 87, wherein L1 is
,
Figure imgf000413_0002
is a bond to the binding agent; and b is an integer from 2 to 8
Figure imgf000414_0001
BA is an antibody;
RN is a hydrogen atom or alkyl;
RM is alkyl; the two bonds represented by are bonds to cysteines of the antibody; and b is an integer from 2 to 8
Figure imgf000414_0002
91. The compound of claim 90, wherein L2 is
or
.
Figure imgf000415_0001
. - rug-conjugate compound of Formula (I):
comprising (a) reacting a deglycosylated antibody or aglycosylated antibody with a compound of Formula (PT1) in the presence of transglutaminase:
Figure imgf000415_0002
wherein:
A is arylene or heteroarylene; and
L is a linker. 93. The method of claim 92 , wherein A is:
,
wherein:
Figure imgf000415_0003
alkenyl, alkynyl, alkoxy, haloalkoxy, aryl, alkaryl, aralkyl, heteroaryl, heteroalkyl,
O
heterocycloalkyl, cyano, nitro, ORA SO2RA RA
, or azido,
wherein RA is alkyl or
Figure imgf000416_0003
n is an integer from 0 to 4;
m is an integer from 0 to 3;
p is an integer from 0 to 6; and q is an integer from 0 to 5.
94. The method of claim 92, wherein the linker is: O S
H
(CH2)b (CH2)b N
wherein:
Figure imgf000416_0001
one is a bond to the payload and the other is a bond to the binding agent; and
Figure imgf000416_0004
Figure imgf000416_0005
b is an integer from 2 to 8. 95. The method of claim 92, wherein the compound of Formula (PT1) is
96. T
Figure imgf000416_0002
.0 and about 8.0 for at least 4 hr. concentration of at least 5 molar equivalents compared to the deglycosylated antibody or aglycosylated antibody. 98. The method of claim 92, wherein the transglutaminase of step (a) is present at 1 to 30 U per milligram of deglycosylated antibody or aglycosylated antibody. 99. The method of any one of claims 92-95, wherein the antibody is deglycosylated with peptide N-glycosidase F (PNGaseF) prior to step (a). 100. The method of any one of claims 92-95, wherein the antibody is aglycosylated. 101. The method of any one of claims 92-97, wherein the compound of Formula (PT1) of step (a) is according to the following formula:
Figure imgf000417_0001
wherein A is:
,
wherein:
Figure imgf000417_0002
R1, independently at each occurrence, is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, aralkyl, halo, haloalkyl, haloalkoxy, heteroaryl, heterocycloalkyl, hydroxyl, cyano, nitro, , o,
wherein
Figure imgf000417_0003
n is an integer from 0 to 4;
m is an integer from 0 to 3; q is an integer from 0 to 5.
102. The method of any one of claims 92-98, wherein the compound of Formula (PT1) of step (a) is:
Figure imgf000418_0001
nducted in one or more solvent(s) selected from the group consisting of water, buffered water, saline water, buffered saline water, and an organic.
104. The method of any one of claims 92-103, wherein step (a) is conducted in water buffered with phosphate, HEPES, or MOPS.
105. The method of any one of claims 92-104, further comprising
(b) reacting the glutaminyl-modified antibody with a reactive payload compound to form an antibody-payload conjugate.
106. The method of any one of claims 92-105, further comprising
(b) reacting the glutaminyl-modified antibody with a reactive linker-payload compound to form an antibody-linker-payload conjugate.
107. The method of any one of claims 92-105, further comprising
(b) reacting the glutaminyl-modified antibody with a reactive linker compound to form an antibody-linker conjugate; and
(c) reacting the antibody-linker conjugate with a reactive payload compound to form an antibody-linker-payload conjugate.
108. A glutaminyl-modified antibody produced by the method of any one of claims 92-104.
109. A pharmaceutical composition comprising the antibody of claim 108 and one or more pharmaceutically acceptable diluents, excipients, or carriers.
110. A method of treating a condition in a subject in need thereof comprising administering to the subject a pharmaceutically acceptable amount of the antibody of claim 108.
1 1 1. The glutaminyl-modified antibody of claim 108 for therapy.
112. The glutaminyl-modified antibody of claim 108 for the treatment of cancer.
113. The compound of claim 27, wherein the compound is selected from
Figure imgf000419_0001
,
,
Figure imgf000420_0001
,
,
Figure imgf000421_0001
, , ,
Figure imgf000422_0001
,
, r
Figure imgf000423_0001
Figure imgf000424_0001
NH
,
Figure imgf000425_0001
.
Figure imgf000426_0001
,
,
Figure imgf000427_0001
Ĭ or
and t s annteger r
Figure imgf000428_0001
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